Methods and apparatus for synthesizing imaging agents, and intermediates thereof

ABSTRACT

The present invention generally relates to methods and system for the synthesis of imaging agents, and precursors thereof. The methods may exhibit improved yields and may allow for the large-scale synthesis of imaging agents, including imaging agents comprising a radioisotope (e.g.,  18 F). Various embodiments of the invention may be useful as sensors, diagnostic tools, and the like. In some cases, methods for evaluating perfusion, including myocardial perfusion, are provided. Synthetic methods of the invention have also been incorporated into an automated synthesis unit to prepare and purify imaging agents that comprise a radioisotope. In some embodiments, the present invention provides a novel methods and systems comprising imaging agent 1, including methods of imaging in a subject comprising administering a composition comprising imaging agent 1 to a subject by injection, infusion, or any other known method, and imaging the area of the subject wherein the event of interest is located.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.provisional application, U.S. Ser. No. 61/302,477, filed Feb. 8, 2010,entitled “Methods and Apparatus for Synthesizing Contrast Agents,Including Radiolabeled Contrast Agents;” U.S. provisional application,U.S. Ser. No. 61/315,376, filed Mar. 18, 2010, entitled “Methods forSynthesizing Contrast Agents and Precursors Thereof;” and U.S.provisional application, U.S. Ser. No. 61/333,693, filed May 11, 2010,entitled “Compositions, Methods, and Systems for Imaging,” each of whichis incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to systems, compositions, methods, andapparatuses for synthesizing imaging agents, and precursors thereof.

BACKGROUND OF THE INVENTION

Mitochondria are membrane-enclosed organelles distributed through thecytosol of most eukaryotic cells. Mitochondria are especiallyconcentrated in myocardial tissue.

Complex 1 (“MC-1”) is a membrane-bound protein complex of 46 dissimilarsubunits. This enzyme complex is one of three energy-transducingcomplexes that constitute the respiratory chain in mammalianmitochondria. This NADH-ubiquinone oxidoreductase is the point of entryfor the majority of electrons that traverse the respiratory chain,eventually resulting in the reduction of oxygen to water (Q. Rev.Biophys. 1992, 25, 253-324). Examples of inhibitors of MC-1 includedeguelin, piericidin A, ubicidin-3, rolliniastatin-1, rolliniastatin-2(bullatacin), capsaicin, pyridaben, fenpyroximate, amytal, MPP+,quinolines, and quinolones (BBA 1998, 1364, 222-235). Studies have shownthat interrupting the normal function of mitochondria couldadvantageously concentrate certain compounds in the mitochondria, andhence in the mitochondria-rich myocardial tissue. Compounds that includean imaging moiety (e.g., ¹⁸F) can be useful in determining such abuild-up of compounds, thereby providing valuable diagnostic markers formyocardial perfusion imaging. In addition, such compounds may findapplication for the diagnosis of coronary artery disease (CAD).

CAD is a major cause of death in modern industrialized countries and ithas been found previously that assessments of regional myocardialperfusion at rest and during stress (exercise or pharmacologic coronaryvasodilation) are valuable for noninvasive diagnosis of CAD. Whilemyocardial perfusion imaging (MPI) with Positron Emission Tomography(PET) has been shown to be superior in some embodiments as compared tosingle photon emission computed tomography (SPECT), widespread clinicaluse of PET MPI has been limited by the previously available PETmyocardial perfusion tracers.

Several PET blood flow tracers, such as rubidium-82 (⁸²Rb) chloride,nitrogen-13 (¹³N) ammonia, and oxygen-15 (¹⁵O) water, have beendeveloped and validated for assessment of myocardial perfusion. ¹³N and¹⁵O are cyclotron-produced isotopes with short half-lives. Therefore,their use is limited to facilities with an on-site cyclotron. Although⁸²Rb is a generator-produced tracer, its short half-life, the high costof the generator, and the inability to perform studies in conjunctionwith treadmill exercise have made this tracer impractical for widespreaduse. Tracers that comprise ¹⁸F have, however, found potentialapplication as imaging agents.

While current methods for preparing compounds comprising an imagingmoiety include [¹⁸F]-fluorination chemistry, many methods focus onnucleophilic [¹⁸F]-fluorination chemistry using potassium fluoride (KF).Characteristically, these methods generate the elemental fluoride sourcethrough anion exchange between, for example, potassium carbonate (K₂CO₃)and a cyclotron-produced [¹⁸F]-containing species, and often requireaddition of the aza-crown ether Kryptofix® 222(4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane) to enhancereactivity. While suitable for production of clinical quantities, themoderate efficiency, demanding purification and complex implementationof such method may not be suitable for widespread commercialapplication.

Accordingly, improved methods, systems, and apparatuses are needed forthe synthesis of imaging agents.

SUMMARY OF THE INVENTION

The invention provides, in a broad sense, methods for synthesizingimaging agents and their precursors, compounds that are imaging agentsprecursors, and methods of use thereof.

In one aspect, in invention provides a method of synthesizing an imagingagent comprising formula:

wherein W is alkyl or heteroalkyl, optionally substituted; R¹ is alkyl,optionally substituted; R² is hydrogen or halide; each R³ can be thesame or different and is alkyl optionally substituted with an imagingmoiety or heteroalkyl optionally substituted with an imaging moiety; andn is 1, 2, 3, 4, or 5; the method comprising steps of: etherification ofprecursor compounds comprising formulae:

wherein n is 1, 2, 3, 4, or 5; R¹ is alkyl, optionally substituted; R²is hydrogen or halide; R³ can be the same or different and are alkyl,heteroalkyl, or a carbonyl-containing group, each optionallysubstituted, R⁵ is hydroxyl or halide; and R⁶ is alkyl, heteroalkyl, ora carbonyl-containing group, each optionally substituted, wherein, whenR⁵ is hydroxyl, at least one of R⁶ and R³ comprises a leaving group; orwherein R⁵ is halide, at least one of R⁶ or R³ comprises a hydroxyl, toproduce a compound comprising formula:

wherein W is alkyl or heteroalkyl, optionally substituted; R¹ is alkyl,optionally substituted; R² is hydrogen or halide; each R³ can be thesame or different and is alkyl optionally substituted with hydroxyl orheteroalkyl optionally substituted with hydroxyl; wherein at least oneR³ comprises hydroxyl; and n is 1, 2, 3, 4, or 5; R¹ is alkyl,optionally substituted; R² is hydrogen or halide; R³ can be the same ordifferent and are alkyl, heteroalkyl, or a carbonyl-containing group,each optionally substituted; reacting a compound comprising formula:

wherein W is alkyl or heteroalkyl, optionally substituted; R¹ is alkyl,optionally substituted; R² is hydrogen or halide; each R³ can be thesame or different and is alkyl optionally substituted with hydroxyl orheteroalkyl optionally substituted with hydroxyl; wherein at least oneR³ comprises hydroxyl; and n is 1, 2, 3, 4, or 5; with asulfonate-containing species to produce a sulfonate-containing compoundcomprising formula:

wherein W is alkyl or heteroalkyl, optionally substituted; R¹ is alkyl,optionally substituted; R² is hydrogen or halide; each R³ can be thesame or different and is alkyl optionally substituted with asulfonate-containing group or heteroalkyl optionally substituted with asulfonate-containing group; wherein at least one R³ comprises asulfonate-containing group; and n is 1, 2, 3, 4, or 5; replacing thesulfonate-containing group of the sulfonate-containing compound with animaging moiety to yield a compound comprising formula:

wherein W is alkyl or heteroalkyl, optionally substituted; R¹ is alkyl,optionally substituted; R² is hydrogen or halide; each R³ can be thesame or different and is alkyl optionally substituted with an imagingmoiety or heteroalkyl optionally substituted with an imaging moiety; andn is 1, 2, 3, 4, or 5; provided that at least one fluorine species ispresent in the compound.

In one aspect, the invention provides a method for ¹⁸F-labeling acompound comprising formula:

wherein R¹ is alkyl; R² is hydrogen or halogen; and R³ is alkylsubstituted with a sulfonate-containing group, alkoxy substituted with asulfonate-containing group, or alkoxyalkyl substituted with asulfonate-containing group. The method comprises reacting the compoundwith an ¹⁸F species in the presence of an ammonium salt or a bicarbonatesalt to form a product comprising the ¹⁸F species.

In some embodiments, R³ is alkoxyalkyl substituted with asulfonate-containing group. In some embodiments, thesulfonate-containing group is mesylate, tosylate, triflate, or1,2-cyclic sulfate. In some embodiments, R² is a halogen. In oneembodiment, R² is chloride. In some embodiments, R¹ is a methyl, ethyl,propyl, n-butyl, s-butyl, or t-butyl. In some embodiments, R¹ ist-butyl. In some embodiments, the product comprises formula:

In another aspect, the invention provides a method for synthesizing aprecursor to (or of) an imaging agent, comprising reacting a compoundcomprising formula (III) with a nucleophile, wherein formula (III)comprises the structure:

wherein W is alkyl or heteroalkyl, optionally substituted; R¹ is alkyl,optionally substituted; R² is hydrogen or halide; each R³ can be thesame or different and is an alkyl optionally substituted with a leavinggroup or heteroalkyl optionally substituted with a leaving group,provided R³ comprises at least one leaving group; and n is 1, 2, 3, 4,or 5, provided at least one R³ is substituted with a leaving group; witha nucleophile wherein the nucleophile replaces the at least one leavinggroup to produce a product (or precursor).

In some embodiments, the nucleophile is ethylene glycol. In someembodiments, reacting the compound with the nucleophile occurs in thepresence of a base. The base may be but is not limited to a metal or ametal salt. The base may be sodium metal, sodium hydride, potassiumt-but oxide, potassium carbonate, or potassium hydroxide. In someembodiments, the base is potassium t-but oxide or potassium hydroxide.In some embodiments, the base is potassium t-but oxide.

In some embodiments, reacting the compound with the nucleophile occursin the presence of a catalyst. The catalyst may be a tetraalkylammoniumiodide including but not limited to a tetraethylammonium iodide.

In some embodiments, the leaving group is a halide including but notlimited to bromide.

In some embodiments, W is —O(CH₂)—; R¹ is t-butyl; R² is chloride; andR³ is alkyl substituted with a leaving group.

In some embodiments, the compound comprising formula (III) comprises thestructure:

In some embodiments, the compound comprising formula (III) comprises thestructure:

In some embodiments, the product (or precursor) comprises formula:

In some embodiments, the product (or precursor) comprises formula:

In some embodiments, the method further comprises reacting a compoundcomprising formula (IV) with a reactant comprising a leaving group toproduce the compound comprising formula (III), wherein formula (IV)comprises the structure:

wherein W is alkyl or heteroalkyl, optionally substituted; R¹ is alkyl,optionally substituted; R² is hydrogen or halide; each R⁴ can be thesame or different and is an alkyl optionally substituted with hydroxylor heteroalkyl optionally substituted with hydroxyl; provided R⁴comprises at least one hydroxyl group; and n is 1, 2, 3, 4, or 5; andwherein the at least one hydroxyl is replaced with the leaving group.

In some embodiments, reacting the compound comprising formula (IV) isperformed in the presence of a halogenation reagent. In someembodiments, the halogenation reagent is a brominating reagent. Thebrominating reagent may be phosphorus tribromide, pyridinium dibromide,or a combination of carbon tetrabromide and triphenylphospine, althoughit is not so limited.

In some embodiments, W is —O(CH₂)—; R¹ is t-butyl; R² is chloride; andR⁴ is alkyl substituted with hydroxyl.

In some embodiments, the compound comprising formula (IV) comprises thestructure:

In some embodiments, the compound comprising formula (IV) comprises thestructure:

In some embodiments, the product comprises formula:

In some embodiments, the product comprises formula:

In some embodiments, the compound comprising formula (IV) is formed byetherification of precursor compounds comprising formulae (IVa) and(IVb):

wherein m is 1, 2, 3, 4, or 5 or greater; R¹ is alkyl, optionallysubstituted; R² is hydrogen or halide; R⁵ is hydroxyl or halide; and R⁶and R⁷ can be the same or different and each is alkyl, heteroalkyl, or acarbonyl-containing group, each of which may be optionally andindependently substituted, wherein when R⁵ is hydroxyl at least one ofR⁶ and R⁷ comprises a leaving group or a group that can be replaced by aleaving group, or when R⁵ is halide, at least one of R⁶ and R⁷ comprisea hydroxyl.

In some embodiments, the compound comprising formula (IV) is formed byetherification of the compounds comprising formulae:

wherein m is 1 or greater; R¹ is alkyl, optionally substituted; R² ishydrogen or halide; R⁵ is hydroxyl or halide; and R⁶ and R⁷ can be thesame or different and each is alkyl, heteroalkyl, or acarbonyl-containing group, any of which may be substituted, wherein whenR⁵ is hydroxyl at least one of R⁶ and R⁷ comprises a leaving group or agroup that can be replaced by a leaving group, or when R⁵ is halide, atleast one of R⁶ and R⁷ comprises a hydroxyl.

In some embodiment, the compound comprising formula (IV) is formed byetherification of precursor compounds comprising formulae (IVa) and(IVd):

wherein R¹ is alkyl, optionally substituted; R² is hydrogen or halide;R⁵ is hydroxyl or halide; and R⁶ and R⁷ can be the same or different andeach is alkyl, heteroalkyl, or a carbonyl-containing group, each ofwhich may be optionally and independently substituted, wherein when R⁵is hydroxyl at least one of R⁶ and R⁷ comprises a leaving group, or whenR⁵ is halide, at least one of R⁶ and R⁷ comprises a hydroxyl.

In some embodiments, the compound comprising formula (IV) is formed byetherification of compounds comprising formulae:

wherein R¹ is alkyl, optionally substituted; R² is hydrogen or halide;R⁵ is hydroxyl or halide; and R⁶ and R⁷ can be the same or different andeach is alkyl, heteroalkyl, or a carbonyl-containing group, any of whichmay be substituted, wherein when R⁵ is hydroxyl at least one of R⁶ andR⁷ comprises a leaving group, or when R⁵ is halide, at least one of R⁶and R⁷ comprises a hydroxyl, or when R⁵ is halide, at least one of R⁶and R⁷ comprises a hydroxyl.

In some embodiments, the etherification comprises reacting the precursorcompounds in the presence of a base. In some embodiments, the basecomprises a carbonate ion.

In some embodiments, R⁵ is halide; and R⁶ and R⁷ is each substitutedalkyl.

In some embodiments, R⁵ is chloride; and R⁶ and R⁷ is each alkylsubstituted with a hydroxyl.

In some embodiments, the compound comprising formula (IV) is synthesizedby etherification of precursor compounds comprising formulae:

wherein R¹ is alkyl, optionally substituted; R² is hydrogen or halide;to form a product comprising formula:

In some embodiments, the compound comprising formula (IV) is synthesizedby etherification of compounds comprising formulae:

to form a product comprising formula:

In some embodiments, R⁵ is hydroxyl; and R⁶ is a carbonyl-containinggroup and R⁷ is a substituted alkyl. In some embodiments, R⁵ ishydroxyl; and R⁶ is an ester and R⁷ is alkyl substituted with a leavinggroup.

In some embodiments, the compound comprising formula (IV) is synthesizedby etherification of the compounds comprising formulae:

to form a product comprising formula:

In some embodiments, the method further comprises exposing the productto a reducing agent to convert the ester group to an alcohol. Thereducing agent may be lithium aluminum hydride, lithium borohydride, ordiisobutylaluminum hydride (DIBAL-H), although it is not so limited.

In another aspect, the invention provides a method for synthesizing animaging agent comprising contacting an imaging agent precursor with afluoride species and an ammonium salt under conditions that result inthe fluoride species replacing the leaving group to produce an imagingagent comprising the fluoride species wherein the molar ratio ofammonium salt to imaging agent precursor is less than 1.5:1, includingabout 1:1 or less.

In some embodiments, the molar ratio of ammonium salt to imaging agentprecursor is about 1:1 or less, or about 0.75:1 or less, or about 0.5:1or less, or about 0.25:1 or less, or about 0.05:1 or less. In someembodiments, the molar ratio of ammonium salt to imaging agent precursoris from about 1:1 to about 0.5:1. In some embodiments, the molar ratioof ammonium salt to imaging agent precursor ranges from about 1.4:1 toabout 0.05:1.

In some embodiments, the ammonium salt is ammonium bicarbonate, ammoniumhydroxide, ammonium acetate, ammonium lactate, ammoniumtrifluoroacetate, ammonium methanesulfonate, ammoniump-toluenesulfonate, ammonium nitrate, ammonium iodide, or ammoniumbisulfate. In some embodiments, the ammonium salt is atetraalkylammonium salt. The ammonium salt may be R₄NHCO₃, wherein R isalkyl. The ammonium salt may be Et₄NHCO₃.

In another aspect, the invention provides a method for synthesizing animaging agent, comprising contacting an imaging agent precursor with afluoride species and a bicarbonate salt under conditions that result inthe fluoride species replacing the leaving group to produce an imagingagent comprising the fluoride species, wherein the molar ratio ofbicarbonate salt to imaging agent precursor is less than 1.5:1,including about 1:1 or less.

In some embodiments, the molar ratio of bicarbonate salt to imagingagent precursor is about 1:1 or less, or about 0.75:1 or less, or about0.5:1 or less, or about 0.25:1 or less, or about 0.05:1. In someembodiments, the molar ratio of bicarbonate salt to imaging agentprecursor is from about 1:1 to about 0.5:1. In some embodiments, themolar ratio of bicarbonate salt to imaging agent precursor ranges fromabout 1.4:1 to about 0.05:1.

In some embodiments, wherein the molar ratio of bicarbonate salt toimaging agent precursor is from about 0.5:1 to about 1:1.

In some embodiments, the bicarbonate salt is a metal bicarbonate. Thebicarbonate salt may be sodium bicarbonate, calcium bicarbonate,potassium bicarbonate, or magnesium bicarbonate, although it is not solimited.

In some embodiments, the bicarbonate salt is ammonium bicarbonate. Insome embodiments, the bicarbonate salt is an tetraalkylammoniumbicarbonate. The bicarbonate salt comprises the formula R₄NHCO₃, whereinR is alkyl. The bicarbonate salt may be Et₄NHCO₃.

In some embodiments, the imaging agent precursor is further exposed to acryptand, such as but not limited to4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane.

In some embodiments, contacting is performed in the absence of acarbonate salt such as but not limited to potassium carbonate.

In some embodiments, the contacting is performed in the absence of acryptand, such as but not limited to4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane.

In another aspect, the invention provides a method for synthesizing animaging agent, comprising contacting an imaging agent precursor with afluoride species under conditions that result in the fluoride speciesreplacing the leaving group to produce an imaging agent comprising thefluoride species, wherein the contacting is performed at a pH below 7.In some embodiments, the contacting is performed at a pH below 6, or ata pH below 5, or at a pH between 5 and 6.

In some embodiments, the leaving group is a sulfonate-containing group.The leaving group may be a mesylate, tosylate, triflate, or 1,2,-cyclicsulfate group. In some embodiments, the leaving group is a tosylategroup. In some embodiments, the fluoride species is an ¹⁸F ion.

In some embodiments, the imaging agent precursor comprises formula (I):

wherein J is selected from the group consisting of N(R²⁸), S, O, C(═O),C(═O)O, NHCH₂CH₂O, a bond, and C(═O)N(R²⁷); when present, K is selectedfrom the group consisting of hydrogen, alkoxyalkyl optionallysubstituted with a leaving group, alkyloxy optionally substituted with aleaving group, aryl optionally substituted with a leaving group, C₁-C₆alkyl optionally substituted with a leaving group, heteroaryl optionallysubstituted with a leaving group, and a leaving group; when present, Lis selected from the group consisting of hydrogen, alkoxyalkyloptionally substituted with a leaving group, alkyloxy optionallysubstituted with a leaving group, aryl optionally substituted with aleaving group, C₁-C₆ alkyl optionally substituted with a leaving group,heteroaryl optionally substituted with a leaving group, and a leavinggroup; M is selected from the group consisting of hydrogen, alkoxyalkyloptionally substituted with a leaving group, alkyloxy optionallysubstituted with a leaving group, aryl optionally substituted with aleaving group, C₁-C₆ alkyl optionally substituted with a leaving group,heteroaryl optionally substituted with a leaving group, and a leavinggroup; or L and M, together with the atom to which they are attached,may form a three-, four-, five-, or six-membered carbocyclic ring; Q ishalo or haloalkyl; n is 0, 1, 2, or 3; R²¹, R²², R²⁷, and R²⁸ areindependently selected from hydrogen, C₁-C₆ alkyl optionally substitutedwith a leaving group, and a leaving group; R²³, R²⁴, R²⁵, and R²⁶ areindependently selected from hydrogen, halogen, hydroxyl, alkyloxy, C₁-C₆alkyl optionally substituted with a leaving group, and a leaving group;R²⁹ is C₁-C₆ alkyl optionally substituted with a leaving group; and Y isselected from the group consisting of a bond, carbon, and oxygen;provided that when Y is a bond, K and L are absent, and M is selectedfrom the group consisting of aryl optionally substituted with a leavinggroup and heteroaryl optionally substituted with a leaving group; andprovided that when Y is oxygen, K and L are absent, and M is selectedfrom hydrogen, alkoxyalkyl optionally substituted with a leaving group,aryl optionally substituted with a leaving group, C₁-C₆ alkyl optionallysubstituted with a leaving group, and heteroaryl optionally substitutedwith a leaving group; provided that at least one leaving group ispresent in formula (I).

In some embodiments, the imaging agent comprises formula (II):

wherein J is selected from the group consisting of N(R²⁸), S, O, C(═O),C(═O)O, NHCH₂CH₂O, a bond, and C(═O)N(R²⁷); when present, K is selectedfrom the group consisting of hydrogen, alkoxyalkyl optionallysubstituted with an imaging moiety, alkyloxy optionally substituted withan imaging moiety, aryl optionally substituted with an imaging moiety,C₁-C₆ alkyl optionally substituted with an imaging moiety, heteroaryloptionally substituted with an imaging moiety, and an imaging moiety;when present, L is selected from the group consisting of hydrogen,alkoxyalkyl optionally substituted with an imaging moiety, alkyloxyoptionally substituted with an imaging moiety, aryl optionallysubstituted with an imaging moiety, C₁-C₆ alkyl optionally substitutedwith an imaging moiety, heteroaryl optionally substituted with animaging moiety, and an imaging moiety; M is selected from the groupconsisting of hydrogen, alkoxyalkyl optionally substituted with animaging moiety, alkyloxy optionally substituted with an imaging moiety,aryl optionally substituted with an imaging moiety, C₁-C₆ alkyloptionally substituted with an imaging moiety, heteroaryl optionallysubstituted with an imaging moiety, and an imaging moiety; or L and M,together with the atom to which they are attached, may form a three- orfour-membered carbocyclic ring; Q is halo or haloalkyl; n is 0, 1, 2, or3; R²¹, R²², R²⁷, and R²⁸ are independently selected from hydrogen,C₁-C₆ alkyl optionally substituted with an imaging moiety, and animaging moiety; R²³, R²⁴, R²⁵, and R²⁶ are independently selected fromhydrogen, halogen, hydroxyl, alkyloxy, C₁-C₆ alkyl optionallysubstituted with an imaging moiety, and an imaging moiety; R²⁹ is C₁-C₆alkyl optionally substituted with an imaging moiety; and Y is selectedfrom the group consisting of a bond, carbon, and oxygen; provided thatwhen Y is a bond, K and L are absent, and M is selected from the groupconsisting of aryl optionally substituted with an imaging moiety andheteroaryl optionally substituted with an imaging moiety; and providedthat when Y is oxygen, K and L are absent, and M is selected fromhydrogen, alkoxyalkyl optionally substituted with an imaging moiety,aryl optionally substituted with an imaging moiety, C₁-C₆ alkyloptionally substituted with an imaging moiety, and heteroaryl optionallysubstituted with an imaging moiety; provided that at least one imagingmoiety is present in formula (II), wherein the imaging moiety is ¹⁸F.

In some embodiments, J is O. In some embodiments, R²⁹ is methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl, each optionallysubstituted with a leaving group. In some embodiments, R²⁹ is t-butyl.In some embodiments, Q is chloro. In some embodiments, each of R²¹, R²²,R²³, R²⁴, R²⁵, R²⁶, and R²⁷ is hydrogen.

In some embodiments, Y is carbon, K and L are hydrogen, and M isselected from the group consisting of alkoxyalkyl optionally substitutedwith a leaving group, alkyloxy optionally substituted with a leavinggroup, aryl optionally substituted with a leaving group, C₁-C₆ alkyloptionally substituted with a leaving group, heteroaryl optionallysubstituted with a leaving group, and a leaving group.

In some embodiments, Y is carbon, K and L is each hydrogen, and M isalkyloxy optionally substituted with a leaving group.

In some embodiments, the imaging agent precursor comprises formula:

wherein L is a leaving group.

In some embodiments, the imaging agent comprises formula:

wherein Im is an imaging moiety.

In some embodiments, the imaging agent precursor comprises:

wherein L is a leaving group.

In some embodiments, the imaging agent comprises formula:

wherein Im is an imaging moiety.

In some embodiments, the imaging agent precursor comprises formula:

In some embodiments, the imaging agent comprising the fluoride speciescomprises formula:

In some embodiments, the method further comprises purifying the imagingagent using at least one purification technique. In some embodiments,the purification technique is chromatography such as but not limited toHPLC. In some embodiments, the purification technique is filtration suchas but not limited to filtration through a C-18 resin.

In some embodiments, the method further comprises combining the imagingagent with a stabilizing agent. In some embodiments, the stabilizingagent is ascorbic acid, or a salt thereof

In another aspect, the invention provides a method for manufacturing animaging agent comprising formula:

the method comprising, (a) contacting a tosylate precursor comprisingformula:

with an anhydrous fluoride species associated with an ammonium salt; (b)heating the mixture of (a); (c) cooling the heated mixture; (d) addingH₂O to the cooled mixture; (e) purifying the mixture from the hydratedmixture of (d) using HPLC with an H₂O/MeCN eluent; and (f) diluting theeluent with a solution of ascorbic acid or a salt thereof.

In some embodiments, step (b) comprises heating the mixture to atemperature between 50° C. and 250° C. In some embodiments, step (b)comprises heating the mixture for less than 5 minutes, less than 10minutes, less than 20, minutes, or less than 30 minutes.

In some embodiments, the method further comprises (g) contacting thediluted eluent of (f) with a C18 resin; (h) washing the contacted C18resin with a solution of ascorbic acid or a salt thereof; (i) eluting

from the C18 resin with absolute ETOH; and (j) diluting the eluent of(i) with a solution or ascorbic acid or a salt thereof.

In some embodiments, the method further comprises (k) asepticallyfiltering the diluted eluent of (j), and (l) optionally, determining thepresence of

in a sample of the aseptic filtrate of (k).

In other aspects, the invention provides imaging agents made by any ofthe preceding methods.

Thus, in one aspect, the invention provides an imaging agent comprisingformula:

wherein the imaging agent is manufactured by (a) contacting a tosylateprecursor comprising formula:

with an anhydrous fluoride species associated with an ammonium salt; (b)heating the mixture of (a); (c) cooling the heated mixture; (d) addingH₂O to the cooled mixture; (e) purifying the mixture from the hydratedmixture of (d) using HPLC with an H₂O/MeCN eluent; and (f) diluting theeluent with a solution of ascorbic acid or a salt thereof.

In some embodiments, step (b) comprises heating the mixture to atemperature between 50° C. and 250° C. In some embodiments, step (b)comprises heating the mixture less than 5 minutes, less than 10 minutes,less than 20, minutes, or less than 30 minutes.

In some embodiments, the manufacturing further comprises (g) contactingthe diluted eluent of (f) with a C18 resin; (h) washing the contactedC18 resin with a solution of ascorbic acid or a salt thereof; (i)eluting

from the C18 resin with absolute ETOH; and (j) diluting the eluent of(i) with a solution of ascorbic acid or a salt thereof.

In some embodiments, the manufacturing further comprises: (k)aseptically filtering the diluted eluent of (j), and (l) optionally,determining the presence of

in a sample of the aseptic filtrate of (k).

In another aspect, the invention provides a method for synthesizing afluorinated compound, comprising reacting, in the presence of acarbonate or bicarbonate, (i) a precursor of the fluorinated compoundcomprising an alkoxyalkyl group substituted with a halide or asulfonate-containing group, with (ii) a salt comprising a fluoridespecies and weakly coordinating cation.

In some embodiments, the alkoxyalkyl group is substituted with asulfonate-containing group. In some embodiments, thesulfonate-containing group is mesylate, tosylate, triflate or 1,2-cyclicsulfate. In some embodiments, the sulfonate-containing group istosylate. In some embodiments, the weakly coordinating cation is atetraalkylammonium cation. In some embodiments, the fluoride species isenriched for ¹⁸F isotope.

In another aspect, the invention provides a method for synthesizing afluorinated compound comprising reacting, in the presence of a carbonateor bicarbonate, (i) a precursor of the fluorinated compound comprisingan alkoxyalkyl substituted with a halide or a sulfonate-containinggroup, with (ii) an ¹⁸F isotope.

In another aspect, the invention provides a method for synthesizing afluorinated compound, comprising reacting (i) a precursor of thefluorinated compound comprising an alkoxyalkyl substituted with a halideor a sulfonate-containing group, with (ii) an ¹⁸F isotope, in thepresence of a tetraalkylammonium bicarbonate or tetraalkylammoniumcarbonate. In some embodiments, the reaction is carried out in thepresence of a tetraalkylammonium bicarbonate.

In some embodiments, the tetraalkylammonium bicarbonate istetraethylammonium bicarbonate, tetrabutylammonium bicarbonate, ortetrahexylammonium bicarbonate.

In another aspect, the invention provides a method for ¹⁸F-labelingcomprising formula:

wherein R is —lower alkyl-sulfonate, R¹ is an C₁-C₁₀ alkyl, and R² is Hor a halogen, comprising reacting the compound with ¹⁸F in the presenceof a tetraalkylammonium bicarbonate or tetraalkylammonium carbonate. Insome embodiments, R is —(CH₂)O(CH₂)_(n)-sulfonate-containing group,wherein n is an integer from 1 to 5. In some embodiments, thesulfonate-containing group is mesylate, tosylate, triflate, or1,2-cyclic sulfate. In some embodiments, R² is a halogen. In someembodiments, R² is chloride. In some embodiments, R¹ is methyl, ethyl,propyl or butyl. In some embodiments, R¹ is t-butyl. In someembodiments, R is —CH₂—O—CH₂—CH₂-tosylate, R¹ is t-butyl and R² ischloride.

In another aspect, the invention provides a method for synthesizing aprecursor to an imaging agent, comprising reacting a compound comprisingformula (V):

wherein W is alkyl or heteroalkyl, optionally substituted; R¹ is alkyl,optionally substituted; and R² is hydrogen or halide; with a nucleophileor a radical species to produce a compound comprising formula (VI):

In some embodiments, the nucleophile is a hydride ion. In someembodiments, the hydride ion is generated from diisobutylaluminumhydride (DIBAL-H). In some embodiments, the radical species is H●.

In some embodiments, the compound comprising formula (V) wherein W is—OCH₂— is synthesized by etherification of precursor compoundscomprising formulae (Va) and (Vb):

to form a product comprising formula:

In some embodiments, R¹ is t-butyl and R² is Cl.

In some embodiments, etherification comprises reacting the precursorcompounds in the presence of a base. In some embodiments, the basecomprises a carbonate ion. In some embodiments, the base comprises ahydroxide ion. In some embodiments, the base is sodium hydroxide ortetramethyl ammonium hydroxide. In some embodiments, the etherificationreaction comprises exposure to sodium hydroxide and benzyltriethylammonium chloride.

In some embodiments, the compound comprising formula (Vb) is produced byexposing the compound comprising formula:

to a reducing agent. In some embodiments, the reducing agent is lithiumaluminum hydride or lithium borohydride. In some embodiments, thereducing agent is lithium aluminum hydride.

In some embodiments, the compound comprising formula:

is produced by reacting methyl 4-formyl benzoate with ethylene glycol inthe presence of an acid.

In another aspect, the invention provides a method for forming asulfonate-containing precursor of an imaging agent, comprising reactinga compound comprising formula:

with a sulfonate-containing species to form a product comprising asulfonate-containing precursor of an imaging agent.

In some embodiments, the sulfonate-containing group is mesylate,tosylate, or triflate. In some embodiments, the sulfonate-containinggroup is tosylate. In some embodiments, the sulfonate-containingprecursor of an imaging agent comprising the formula:

In some cases, the sulfonate-containing precursor is reacted with animaging moiety to form an imaging agent.

In some embodiments, the imaging moiety is a radioisotope. In someembodiments, the imaging moiety is ¹¹C, ¹³N, ¹⁸F, ¹²³I, ¹²⁵I, ^(99m)Tc,⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, or ⁶⁸Ga. In some embodiments, the imagingmoiety is ¹⁸F.

In some embodiments, the imaging agent has the structure:

In another aspect, the invention provides a method for synthesizing animaging agent, comprising reacting precursor compounds comprisingformulae:

via an etherification reaction to form a first compound comprising theformula:

exposing the first compound to a reducing agent to form a secondcompound comprising a benzylic alcohol; treating the second compoundwith phosphorus tribromide to form a third compound comprising abenzylic bromide; reacting the third compound with ethylene glycol toproduce a fourth compound comprising the formula:

and reacting the fourth compound with a sulfonate-containing species toform a product comprising a sulfonate-containing precursor of an imagingagent. In some cases, the method further comprises reacting thesulfonate-containing precursor of an imaging agent with an imagingmoiety to form the imaging agent.

In another aspect, the invention provides a compound having thestructure:

wherein the compound is synthesized using any of the preceding methods.

In another aspect, the invention provides a compound comprising formula:

In another aspect, the invention provides a compound comprising formula:

In another aspect, the invention provides a compound comprising formula:

In another aspect, the invention provides a compound comprising formula:

In another aspect, the invention provides a method of imaging a subject,comprising administering to a subject a first dose of imaging agentcomprising the formula:

in an amount between about 1 mCi and about 4 mCi; acquiring at least onefirst image of a portion of the subject; subjecting the subject tostress; administering to the subject undergoing stress a second dose ofthe imaging agent in an amount greater than the first dose of theimaging agent by at least about 1.5 times the first dose of the imagingagent; and acquiring at least one second image of the portion of thesubject.

In some embodiments, the second dose of the imaging agent isadministered within less than about 48 hours, 24 hours, 18 hours, 12hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes,or 15 minutes after acquiring the at least one first image. In someembodiments, the second dose of the imaging agent is at least 2.0 timesgreater than the first dose of the imaging agent. In some embodiments,the first image is obtained during an image acquisition period between 1and 20 minutes. In some embodiments, the second image is obtained duringan image acquisition period between 1 and 20 minutes. In someembodiments, the portion of the subject is at least a portion of thecardiovascular system. In some embodiments, the portion of thecardiovascular system is at least a portion of the heart. In someembodiments, the acquiring employs positron emission tomography.

In some embodiments, the method further comprises determining thepresence or absence of a cardiovascular disease or condition in thesubject. In some embodiments, the cardiovascular disease is coronaryartery disease or myocardial ischemia.

In some embodiments, the imaging agent is administered as a formulationcomprising water, less than about 5% ethanol, and less than about 50mg/mL sodium ascorbate. In some embodiments, the formulation comprisingthe imaging agent is administered via an intravenous bolus injection. Insome embodiments, the stress is induced by exercising the subject. Insome embodiments, the second dose of the imaging agent is administeredduring the exercise.

In some embodiments, the first dose of the imaging agent is betweenabout 1.0 mCi to about 2.5 mCi. In some embodiments, the first dose ofthe imaging agent is between about 1.7 mCi to about 2.0 mCi. In someembodiments, the first dose of the imaging agent is between about 2.5 toabout 3.0 mCi.

In some embodiments, the wait time between acquiring at least one firstimage of a portion of the subject and administering to the subject asecond dose of the imaging agent is 60 minutes. In some embodiments, thesecond dose of the imaging agent is administered in an amount that is atleast 2.5, or at least 3.0 times greater than the first dose of theimaging agent. In some embodiments, the second dose of the imaging agentis administered in an amount between 2.5 and about 5.0, or 2.5 and 4.0,or 3.0 and 4.0 time greater, or between 3.0 and 5.0 times greater thanthe first dose of the imaging agent. In some embodiments, the seconddose of the imaging agent is between about 8.6 mCi and about 9.0 mCi, orbetween about 8.6 mCi and about 9.5 mCi, or between about 9.0 to about9.5 mCi.

In some embodiments, the stress is pharmacological stress. In someembodiments, the pharmacological stress is induced by administering apharmacological stress agent to the subject. In some embodiments, thepharmacological stress agent is a vasodilator. In some embodiments, thevasodilator is adenosine. In some embodiments, the second dose of theimaging agent is administered after the subject has been administeredthe pharmacological stress agent. In some embodiments, the second doseof the imaging agent is administered when the subject is at peakvasodilation from the pharmacological stress agent.

In some embodiments, the first dose of the imaging agent is betweenabout 2.0 mCi to about 3.5 mCi. In some embodiments, the first dose ofthe imaging agent is between about 2.4 mCi to about 3.0 mCi or betweenabout 2.4 mCi to about 2.9 mCi. In some embodiments, the first dose ofthe imaging agent is between about 2.5 mCi to about 3.0 mCi or betweenabout 2.5 mCi and about 3.5 mCi.

In some embodiments, the wait time between acquiring at least one firstimage of a portion of the subject and administering to the subject asecond dose of the imaging agent is 30 minutes. In some embodiments, thesecond dose of the imaging agent is administered in an amount at least2.0 times greater than the first dose of the imaging agent. In someembodiments, the second dose of the imaging agent is administered in anamount that is between 2 to 3 times greater than the first dose of theimaging agent, including 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, or 2.9times greater.

In some embodiments, the second dose of the imaging agent is betweenabout 5.7 mCi and about 6.2 mCi. In some embodiments, the second dose ofthe imaging agent is between about 6.0 mCi and about 6.5 mCi or betweenabout 5.7 mCi and about 6.5 mCi. In some embodiments, the total of thefirst and second dose of the imaging agent does not exceed about 14 mCi.

In another aspect, the invention provides a syringe comprising acomposition comprising the imaging agent comprises the formula:

wherein the syringe adsorbs less than 20%, 19%, 18%, 17%, 16%, 15%, 14%,13,% 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of theimaging agent. In some cases, the syringe adsorbs between about 1% andabout 20%, or between about 5% and about 15%, or between about 1% andabout 15%, or between 2% and about 10%, or between about 5% and about20%.

In some embodiments, the syringe comprises a plunger that adsorbs lessthan 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13,% 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the imaging agent. In someembodiments, the syringe comprises a plunger that is not rubber-tipped.In some embodiments, the syringe is a latex-free syringe. In someembodiments, the syringe comprises no rubber, and no silicon lubricants.In some embodiments, the syringe is a non-reactive syringe. In somecases, the syringe adsorbs between about 1% and about 20%, or betweenabout 5% and about 15%, or between about 1% and about 15%, or between 2%and about 10%, or between about 5% and about 20%.

In some embodiments, the syringe further comprises sodium ascorbate,ethanol, and water. In some embodiments, the imaging agent is in asolution comprising less than 4% ethanol and less than 50 mg/mL sodiumascorbate in water.

In some embodiments, the imaging agent is present in the syringe in adose between about 1.5 and about 14 mCi.

In another aspect, the invention provides a method of imaging a subject,comprising subjecting a subject to stress; administering to the subjecta first dose of an imaging agent comprising the formula:

in an amount between about 1 mCi and about 4 mCi; acquiring at least onefirst image of a portion of the subject; administering to the subject asecond dose of the imaging agent in an amount greater than the firstdose of the imaging agent; and acquiring at least one second image ofthe portion of the subject.

In some embodiments, the amount of the second dose is more than 1.5times the amount of the first dose.

In another aspect, the invention provides a method of imaging a subject,comprising subjecting a subject to stress; administering to the subjecta dose of an imaging agent comprising formula:

in an amount less 20 mCi; and acquiring at least one first image of aportion of the subject.

In some embodiments, the amount of the dose is less than 14 mCi. In someembodiments, the amount of the dose is between 1 mCi and 4 mCi.

In another aspect, the invention provides a cassette for the preparationof an imaging agent comprising formula:

comprising: (i) a vessel containing an imaging agent precursorcomprising formula:

and (ii) a conduit for adding a source of ¹⁸F.

In another aspect, the invention provides an automated reaction system,comprising:

the foregoing cassette. In another aspect, the invention provides anapparatus for synthesizing an imaging agent comprising a lineararrangement of a plurality of stopcock manifolds connected one or moreof the components selected from the group consisting of a [¹⁸O]H₂Orecovery system, gas inlet, reservoir with solution of imaging agentprecursor, vial, anion exchange cartridge, C-18 cartridge, syringe,solvent reservoir, reaction vessel, HPLC system, collection vessel,reservoir for solution of ascorbic acid or salt thereof, and exhaustoutlet.

In some embodiments, the apparatus further comprising tubing. In someembodiment, the apparatus further comprising an imaging agent synthesismodule, wherein the apparatus is fluidically connected to the apparatus.In some embodiments, the apparatus is capable of carrying out the methodas described herein. In some embodiments, the apparatus is capable ofpreparing an imaging agent comprising formula:

In some embodiments, the invention provides an apparatus comprising thecomponents arranged as shown in FIG. 8. In some cases, the componentsare arranged in the order: (1) gas inlet; (2) [¹⁸O]H₂O recovery system;(3) anion exchange cartridge; (4) MeCN reservoir; (5) syringe; (6)reservoir with solution of imaging agent precursor; (7) reaction vessel;(8) HPLC system; (9) reservoir with solution of ascorbic acid or a saltthereof; (10) collection vessel; (11) ethanol reservoir; (12) vial withfinal product; (13) Sep-pack cartridge; (14) reservoir with solution ofascorbic acid or a salt thereof; (15) reaction vessel; and (16) exhaustoutlet.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of a nucleophilic [¹⁸F]-fluorination reactionusing an imaging agent precursor and a fluoride source to form animaging agent.

FIG. 2 shows various reaction pathways of an imaging agent precursorduring a nucleophilic fluorination reaction.

FIG. 3 shows an exemplary synthesis of an intermediate compound.

FIG. 4 shows an alternative synthesis of an intermediate compound.

FIG. 5 shows another alternative synthesis of an intermediate compound.

FIG. 6 shows a flow chart describing a method for synthesizing animaging agent.

FIG. 7 is a schematic representation of a system for synthesizing animaging agent using a modified Explora GN synthesis module.

FIG. 8 is a schematic representation of a cassette, with associatedcolumns and reagents for synthesizing an imaging agent using a modifiedGE-Tracerlab-MX synthesis module.

FIGS. 9A-C includes (A) a graph illustrating the changes in productdistribution as a function of molar concentration of bicarbonate salt,(B) a graph illustrating the product distribution as a function ofreaction time, and (C) a graph illustrating the changes in productdistribution as a function of molar concentration of imaging agentprecursor.

FIG. 10 illustrates non-limiting examples of imaging agents which may beprepared using the fluorination methods as described herein, in someembodiments.

FIG. 11 shows whole body coronal sections at the level of the myocardiumfrom a representative human subject at different time points afteradministration of imaging agent 1.

FIGS. 12A-C shows representative cardiac images of imaging agent 1 incontrol rabbits in cardiac short- and long-axis views, and polar maps.

FIGS. 12D-F shows representative cardiac images of imaging agent 1 inchronic myocardial infarct (MI) rabbits in cardiac short- and long-axisviews, and polar maps.

FIG. 13 shows a plot of reader's scores versus percentage decreases fromthe maximum value for rest image data of a study followingadministration of imaging agent 1 injection to subjects, according to anon-limiting embodiment.

Other aspects, embodiments, and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying figures areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patentsincorporated herein by reference are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention generally relates to systems, compositions,cassettes, methods, and apparatuses for the synthesis of imaging agentsand precursors thereof. In some aspects the invention relates to imagingagents synthesized using methods described herein.

In some embodiments, the present invention relates to methods forsynthesizing an imaging agent, for example, by reacting an imaging agentprecursor with a source of an imaging moiety. As described herein, insome cases, the method involves the use of one or more additives (e.g.,salts) that may facilitate a chemical reaction. The methods may exhibitimproved yields and may allow for the widespread synthesis of imagingagents, including imaging agents comprising a radioisotope (e.g., ¹⁸F).The imaging agents may be useful as sensors, diagnostic tools, and thelike. Synthetic methods for preparing an imaging agent have also beendesigned to use an automated synthesis system to prepare and purifyimaging agents that comprise a radioisotope. In some aspects, theinvention allows radiolabeled imaging agents to be made using anucleophilic reaction system, including, but not limited to, the ExploraGN or RN synthesis system (Siemens Medical Solutions USA, Inc.),GE-Tracerlab-MX synthesis system (GE Healthcare), Eckert & ZeiglerModular-Lab Synthesis system, etc., which are commonly available at PETmanufacturing facilities (PMF).

In some embodiments, the present invention provides methods forsynthesizing an imaging agent precursor, wherein the imaging agentprecursor is reacted with a source of an imaging moiety to form theimaging agent. As will be understood by those of ordinary skill in theart, it is advantageous to utilize methods which involve high-yieldingreactions and a relatively low number of synthetic and/or purificationsteps. Accordingly, many of the methods provided herein for synthesizingan imaging agent precursor provide the imaging agent precursor in fewersteps than previously reported, with greater ease of synthesis and/or ata higher yield.

In some embodiments, the present invention provides methods of imaging,including methods of imaging in a subject that includes administering acomposition or formulation (e.g., that comprises imaging agent 1, asdescribed herein) to the subject by injection, infusion, or any methodof administration, and imaging a region of the subject that is ofinterest. Regions of interest may include, but are not limited to, theheart, cardiovascular system, cardiac vessels, blood vessels (e.g.,arteries, veins), brain, and other organs. A parameter of interest, suchas blood flow, cardiac wall motion, or perfusion, can be imaged anddetected using methods and/or systems of the invention. In some cases,methods for evaluating perfusion, including myocardial perfusion, areprovided.

As used herein, the term “imaging agent” refers to any species thatincludes at least one atom, or group of atoms, that may generate adetectable signal itself, or upon exposure to an external source ofenergy (e.g., electromagnetic radiation, ultrasound, etc.). Typically,the imaging agent may be administered to a subject in order to provideinformation relating to at least a portion of the subject (e.g., human).In some cases, an imaging agent may be used to highlight a specific areaof a subject, rendering organs, blood vessels, tissues, and/or otherportions more detectable and more clearly imaged. By increasing thedetectability and/or image quality of the object being studied, thepresence and extent of disease and/or injury can be determined. Theimaging agent may include a radioisotope for nuclear medicine imaging. Anon-liming example of an imaging agent, also referred herein as imagingagent 1, comprises the formula:

As used herein, an “imaging moiety” refers to an atom or group of atomsthat is capable of producing a detectable signal itself or upon exposureto an external source of energy (e.g., imaging agents comprising imagingmoieties may allow for the detection, imaging, and/or monitoring of thepresence and/or progression of a condition), pathological disorder,and/or disease. Nuclear medicine imaging agents can include ¹¹C, ¹³N,¹⁸F, ¹²³I, ¹²⁵I, ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, and ⁶⁸Ga asthe imaging moiety. In some embodiments, the imaging moiety is ¹⁸F.Imaging agents based on ¹⁸F have been used for imaging hypoxia andcancer (Drugs of the Future 2002, 27, 655-667).

In some embodiments, a compound (e.g., an imaging agent, a fluoridespecies) may be isotopically-enriched with fluorine-18.“Isotopically-enriched” refers to a composition containing isotopes ofan element such that the resultant isotopic composition is other thanthe natural isotopic composition of that element. With regard to thecompounds provided herein, when a particular atomic position isdesignated as ¹⁸F, it is to be understood that the abundance of ¹⁸F atthat position is substantially greater than the natural abundance of¹⁸F, which is essentially zero. In some embodiments, a fluorinedesignated as ¹⁸F may have a minimum isotopic enrichment factor of about0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%,about 0.5%, about 0.75%, about 1%, about 2%, about 3%, about 4%, about5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, or greater. Theisotopic enrichment of the compounds provided herein can be determinedusing conventional analytical methods known to one of ordinary skill inthe art, including mass spectrometry and HPLC.

Exemplary Methods for Synthesizing Imaging Agents

The present invention provides methods for synthesizing imaging agents.In some cases, the imaging agent is formed by reacting an imaging agentprecursor with an imaging moiety. In certain embodiments, a methodinvolves reacting between an imaging agent precursor comprising aleaving group with a source of an imaging moiety (e.g., a fluoridespecies).

For example, the imaging moiety replaces the leaving group via asubstitution reaction, such as an S_(N)2 or S_(N)1 reaction. That is,during the reaction an imaging moiety replaces the leaving group,thereby producing the imaging agent.

The methods described herein may be used for the synthesis of a widevariety of imaging agents from an imaging agent precursor. Generally,the imaging agent precursor may include at least one leaving group thatmay be displaced by an imaging moiety, such as an ¹⁸F species. Imagingagent precursors may be synthesized using methods known to those ofordinary skill in the art and as described below.

In some embodiments, the imaging agent precursor comprises formula (I):

wherein:

J is selected from the group consisting of N(R²⁸), S, O, C(═O), C(═O)O,NHCH₂CH₂O, a bond, and C(═O)N(R²⁷);

when present, K is selected from the group consisting of hydrogen,alkoxyalkyl optionally substituted with a leaving group, alkyloxyoptionally substituted with a leaving group, aryl optionally substitutedwith a leaving group, C₁-C₆ alkyl optionally substituted with a leavinggroup, heteroaryl optionally substituted with a leaving group, and aleaving group;

when present, L is selected from the group consisting of hydrogen,alkoxyalkyl optionally substituted with a leaving group, alkyloxyoptionally substituted with a leaving group, aryl optionally substitutedwith a leaving group, C₁-C₆ alkyl optionally substituted with a leavinggroup, heteroaryl optionally substituted with a leaving group, and aleaving group;

M is selected from the group consisting of hydrogen, alkoxyalkyloptionally substituted with a leaving group, alkyloxy optionallysubstituted with a leaving group, aryl optionally substituted with aleaving group, C₁-C₆ alkyl optionally substituted with a leaving group,heteroaryl optionally substituted with a leaving group, and a leavinggroup; or

L and M, together with the atom to which they are attached, may form athree-, four-, five-, or six-membered carbocyclic ring;

Q is halo or haloalkyl;

n is 0, 1, 2, or 3;

R²¹, R²², R²⁷, and R²⁸ are independently selected from hydrogen, C₁-C₆alkyl optionally substituted with a leaving group, and a leaving group;

R²³, R²⁴, R²⁵, and R²⁶ are independently selected from hydrogen,halogen, hydroxyl, alkyloxy, C₁-C₆ alkyl optionally substituted with aleaving group, and a leaving group;

R²⁹ is C₁-C₆ alkyl optionally substituted with a leaving group; and

Y is selected from the group consisting of a bond, carbon, and oxygen;provided that when Y is a bond, K and L are absent, and M is selectedfrom the group consisting of aryl optionally substituted with a leavinggroup and heteroaryl optionally substituted with a leaving group; andprovided that when Y is oxygen, K and L are absent, and M is selectedfrom hydrogen, alkoxyalkyl optionally substituted with a leaving group,aryl optionally substituted with a leaving group, C₁-C₆ alkyl optionallysubstituted with a leaving group, and heteroaryl optionally substitutedwith a leaving group;

provided that at least one leaving group is present in formula (I).

In some embodiments, a method of the present invention comprisespreparing an imaging agent comprising formula (II):

wherein:

J is selected from the group consisting of N(R²⁸), S, O, C(═O), C(═O)O,NHCH₂CH₂O, a bond, and C(═O)N(R²⁷);

when present, K is selected from the group consisting of hydrogen,alkoxyalkyl optionally substituted with an imaging moiety, alkyloxyoptionally substituted with an imaging moiety, aryl optionallysubstituted with an imaging moiety, C₁-C₆ alkyl optionally substitutedwith an imaging moiety, heteroaryl optionally substituted with animaging moiety, and an imaging moiety;

when present, L is selected from the group consisting of hydrogen,alkoxyalkyl optionally substituted with an imaging moiety, alkyloxyoptionally substituted with an imaging moiety, aryl optionallysubstituted with an imaging moiety, C₁-C₆ alkyl optionally substitutedwith an imaging moiety, heteroaryl optionally substituted with animaging moiety, and an imaging moiety;

M is selected from the group consisting of hydrogen, alkoxyalkyloptionally substituted with an imaging moiety, alkyloxy optionallysubstituted with an imaging moiety, aryl optionally substituted with animaging moiety, C₁-C₆ alkyl optionally substituted with an imagingmoiety, heteroaryl optionally substituted with an imaging moiety, and animaging moiety; or

L and M, together with the atom to which they are attached, may form athree-, four-, five-, or six-membered carbocyclic ring;

Q is halo or haloalkyl;

n is 0, 1, 2, or 3;

R²¹, R²², R²⁷, and R²⁸ are independently selected from hydrogen, C₁-C₆alkyl optionally substituted with an imaging moiety, and an imagingmoiety;

R²³, R²⁴, R²⁵, and R²⁶ are independently selected from hydrogen,halogen, hydroxyl, alkyloxy, C₁-C₆ alkyl optionally substituted with animaging moiety, and an imaging moiety;

R²⁹ is C₁-C₆ alkyl optionally substituted with an imaging moiety; and

Y is selected from the group consisting of a bond, carbon, and oxygen;provided that when Y is a bond, K and L are absent, and M is selectedfrom the group consisting of aryl optionally substituted with an imagingmoiety and heteroaryl optionally substituted with an imaging moiety; andprovided that when Y is oxygen, K and L are absent, and M is selectedfrom hydrogen, alkoxyalkyl optionally substituted with an imagingmoiety, aryl optionally substituted with an imaging moiety, C₁-C₆ alkyloptionally substituted with an imaging moiety, and heteroaryl optionallysubstituted with an imaging moiety;

provided that at least one imaging moiety is present in formula (II).That is, the imaging agent comprising formula (II) is formed from animaging agent precursor comprising formula (I), wherein a leaving groupof the imaging agent precursor comprising formula (I) is replaced by animaging moiety. In some embodiments, the imaging moiety is ¹⁸F.

In some cases, J is selected from N(R²⁷), S, O, C(═O), C(═O)O,NHCH₂CH₂O, a bond, or C(═O)N(R²⁷). In some cases when present, K isselected from hydrogen, alkoxyalkyl optionally substituted with aleaving group, alkyloxy, aryl, C₁-C₆ alkyl optionally substituted with aleaving group, heteroaryl, and a leaving group. In some cases, whenpresent, L is selected from hydrogen, alkoxyalkyl optionally substitutedwith a leaving group, alkyloxy, aryl, C₁-C₆ alkyl optionally substitutedwith a leaving group, heteroaryl, and a leaving group. In some case, Mis selected from hydrogen, alkoxyalkyl optionally substituted with aleaving group, alkyloxy, aryl, C₁-C₆ alkyl optionally substituted with aleaving group, heteroaryl, and a leaving group. In some cases, L and M,together with the atom to which they are attached, form a three- orfour-membered carbocyclic ring. In some cases Q is halo or haloalkyl. Insome cases, n is 0, 1, 2, or 3. In some cases, R²¹, R²², R²³, R²⁴, R²⁵,R²⁶, and R²⁷ are independently selected from hydrogen, C₁-C₆ alkyloptionally substituted with a leaving group, and a leaving group. Insome cases R²⁹ is C₁-C₆ alkyl optionally substituted with a leavinggroup. In some cases, Y is selected from a bond, carbon, and oxygen;provided that when Y is a bond, K and L are absent and M is selectedfrom aryl and heteroaryl; and provided that when Y is oxygen, K and Lare absent and M is selected from hydrogen, alkoxyalkyl optionallysubstituted with a leaving group, aryl, C₁-C₆ alkyl optionallysubstituted with a leaving group, and heteroaryl.

In some cases, J is O. In some cases R²⁹ is methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, or t-butyl, each may be optionallysubstituted with a leaving group. In certain embodiment, R²⁹ is t-butyl.In some cases, Q is chloro. In some cases, all of R²¹, R²², R²³, R²⁴,R²⁵, R²⁶ and R²⁷ are hydrogen. In some cases, Y is carbon, K and L arehydrogen, and M is alkoxyalkyl optionally substituted with a leavinggroup, alkyloxy optionally substituted with a leaving group, aryloptionally substituted with a leaving group, C₁-C₆ alkyl optionallysubstituted with a leaving group, heteroaryl optionally substituted witha leaving group, or a leaving group. In some cases, Y is carbon, K and Lare hydrogen, and M is alkyloxy optionally substituted with a leavinggroup.

In some embodiments, the imaging agent precursor comprises the formula:

wherein R²¹, R²², R²⁹, Q, J, and n are as described herein, and L is aleaving group.

In some embodiments, the imaging agent comprises the formula:

wherein R²¹, R²², R²⁹, Q, J, and n are as described herein, and I_(m) isan imaging moiety.

In some embodiments, the imaging agent precursor comprises the formula:

wherein R²⁹ and Q are as described herein, and L is a leaving group.

In some embodiments, the imaging agent comprises the formula:

wherein R²⁹ and Q are as described herein, and Im is an imaging moiety.

In one set of embodiments, the imaging agent precursor comprises theformula:

herein referred to as imaging agent precursor 1 (see FIG. 1).

In some cases, the imaging agent comprises the formula:

herein referred to as imaging agent 1 (see FIG. 1).

Other non-limiting examples of imaging agents that may be prepared usinga fluorination methods of the present invention are shown in FIG. 10. Insome cases, the imaging agent precursor is not a salt.

Various methods may be used to synthesize an imaging agent precursor ofthe formula (I), including an etherification reaction (e.g., Mitsonobureaction) between two alcohols, or between a phenol and an alcohol. Insome cases, a leaving group may be installed by conversion of a hydroxylgroup into a tosylate group or other leaving group, for example, byreaction with p-toluenesulfonate chloride in the presence of a base(e.g., DMAP). Additional methods for the synthesis of an imaging agenthaving the structure comprising formula (II) or an imaging agentprecursor having the structure comprising formula (I) are described inInternational Publication No. WO2005/079391, the contents of which areincorporated herein by reference.

In some embodiments, a method for synthesizing an imaging agentcomprises contacting an imaging agent precursor (e.g., a compoundcomprising formula (I)) with a fluoride species and an ammonium saltunder conditions that result in the fluoride species replacing theleaving group to produce an imaging agent (e.g., a compound comprisingformula (II)) comprising the fluorine species wherein the molar ratio ofammonium salt to imaging agent precursor is less than about 1.5:1, orabout 1:1 or less (or any ratio described herein).

In some embodiments, a method for synthesizing an imaging agentcomprises contacting an imaging agent precursor (e.g., a compoundcomprising formula (I)) with a fluoride species and a bicarbonate saltunder conditions that result in the fluoride species replacing theleaving group to produce an imaging agent (e.g., a compound comprisingformula (II)) comprising the fluorine species, wherein the molar ratioof bicarbonate salt to imaging agent precursor is less than about 1.5:1,or is about 1:1 or less (or any ratio described herein).

In some embodiments, a method for synthesizing an imaging agentcomprises contacting an imaging agent precursor (e.g., a compoundcomprising formula (I)) with a fluoride species under conditions thatresult in the fluoride species replacing the leaving group to produce animaging agent (e.g., a compound comprising formula (II)) comprising thefluorine species, wherein the contacting is performed at a pH below 7.

In some embodiments, a method for ¹⁸F-labeling a compound comprising theformula:

wherein:

R¹ is alkyl, optionally substituted;

R² is hydrogen or halogen; and

R³ is alkyl substituted with a sulfonate-containing group, alkoxysubstituted with a sulfonate-containing group, or alkoxyalkylsubstituted with a sulfonate-containing group, comprises reacting thecompound with an ¹⁸F species in the presence of an ammonium salt or abicarbonate salt to form a product comprising the ¹⁸F species.

In some embodiments, a method for manufacturing an imaging agentcomprising the formula:

comprises

(a) contacting a tosylate precursor comprising the formula:

with a fluoride species associated with an ammonium salt;

(b) heating the mixture of (a);

(c) cooling the heated mixture;

(d) adding H₂O to the cooled mixture;

(e) purifying the mixture from the hydrated mixture of (d) using HPLCwith an H₂O/MeCN eluent; and

(f) diluting the eluent with a solution of ascorbic acid or a saltthereof.

In some cases, step (b) comprises heating the mixture to a temperaturebetween 50° C. and 250° C. In some cases, the heating step (b) comprisesheating the mixture for less than 5 minutes, less than 10 minutes, lessthan 20, minutes, or less than 30 minutes. In some cases, the methodfurther comprises:

(g) contacting the diluted eluent of (f) with a C18 resin;

(h) washing the contacted C18 resin with a solution of ascorbic acid ora salt thereof;

(i) eluting

from the C18 resin with absolute ethanol; and

(j) diluting the eluent of (i) with a solution of ascorbic acid or asalt thereof (e.g., sodium salt).

In some cases, the method further comprises

(k) aseptically filtering the diluted eluent of (j), and

(l) optionally, determining the presence of

in a sample of the aseptic filtrate of (k).

In some embodiments, an imaging agent comprising the formula:

is manufactured by:

(a) contacting a tosylate precursor comprising the formula:

with an anhydrous fluoride species associated with an ammonium salt;

(b) heating the mixture of (a);

(c) cooling the heated mixture;

(d) adding H₂O to the cooled mixture;

(e) purifying the mixture from the hydrated mixture of (d) using HPLCwith an H₂O/MeCN eluent; and

(f) diluting the eluent with a solution of ascorbic acid or a saltthereof.

In some cases, step (b) comprises heating the mixture to a temperaturebetween 50° C. and 250° C. In some cases, the heating step (b) comprisesheating the mixture less than 5 minutes, less than 10 minutes, less than20, minutes, or less than 30 minutes. In some cases, the manufacturingfurther comprises:

(g) contacting the diluted eluent of (f) with a C18 resin;

(h) washing the contacted C18 resin with a solution of ascorbic acid ora salt thereof;

(i) eluting

from the C18 resin with absolute ethanol; and

(j) diluting the eluent of (i) with a solution of ascorbic acid or asalt thereof.

In some cases, the manufacturing further comprises:

(k) aseptically filtering the diluted eluent of (j), and

(l) optionally, determining the presence of

in a sample of the aseptic filtrate of (k).

In some embodiments, a method for synthesizing a fluorinated compoundcomprises reacting, in the presences of a carbonate or bicarbonate ion,(i) a precursor of the fluorinated compound comprising an alkoxyalkylgroup substituted with a halide or a sulfonate-containing group, with(ii) a salt comprising a fluoride species and weakly coordinatingcation.

As used herein, the term “leaving group” is given its ordinary meaningin the art of synthetic organic chemistry and refers to an atom or agroup capable of being displaced by a nucleophile. Examples of suitableleaving groups include, but are not limited to, halides (such aschloride, bromide, or iodide), alkoxycarbonyloxy, aryloxycarbonyloxy,alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy),arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl,haloformates, and the like. In some cases, the leaving group is asulfonic acid ester, such as toluenesulfonate (tosylate, TsO),methanesulfonate (mesylate, MsO), or trifluoromethanesulfonate(triflate, TfO). In some cases, the leaving group may be a brosylate,such as p-bromobenzenesulfonyl. In some cases, the leaving group may bea nosylate, such as 2-nitrobenzenesulfonyl. The leaving group may alsobe a phosphineoxide (e.g., formed during a Mitsunobu reaction) or aninternal leaving group such as an epoxide or cyclic sulfate. In someembodiments, the leaving group is a sulfonate-containing group. In someembodiments, the leaving group is a tosylate group.

In certain embodiments, the invention provides methods of synthesizingan imaging agent comprising a halogen. For example, the method mayinvolve a halogenation reaction. In some embodiments, methods forsynthesizing an imaging agent comprising a fluoride (e.g., enriched with¹⁸F) are provided. The method comprises contacting an imaging agentprecursor with a source of a fluoride under conditions that result inthe fluoride replacing a leaving group of the precursor to produce animaging agent comprising a fluoride species. In certain embodiments, themethod involves a nucleophilic fluorination reaction. That is, animaging agent precursor comprising a leaving group is reacted in thepresence of a fluoride species, whereby S_(N)2 or S_(N)1 displacement ofthe leaving group by the fluoride species produces the imaging agent. Insome embodiments, the fluoride species is enriched with ¹⁸F. FIG. 1shows an illustrative example, where imaging agent precursor 1 istreated with an ¹⁸F species to produce imaging agent 1 via asubstitution reaction.

In some embodiments, one or more additives may be incorporated into thereaction mixture of the imaging agent precursor and the fluoridespecies. The additive may, in some cases, facilitate reaction betweenthe imaging agent precursor and the fluoride species and/or may aid instabilizing the imaging agent. For example, the fluoride species mayhave relatively low reactivity (e.g., nucleophilicity), and addition ofan additive may enhance the reactivity of the fluoride species. As anillustrative embodiment, a fluorine species may be a negatively chargedfluoride ion (e.g., an isotopically-enriched ¹⁸F ion), and an additivemay be used to bind to any positively charged counterions present withinthe reaction mixture, thereby enhancing the reactivity of the fluorideion. In some embodiments, the additives may decrease the rate ofundesired side reactions, as described below.

In some cases, the additive may be combined with the fluoride speciesprior to contact with the imaging agent precursor. For example, incertain embodiments a solution comprising the fluoride species and theadditive is prepared, and the solution is added to the imaging agentprecursor. In other embodiments, a solid comprising the fluoride speciesand the additive is prepared, and the solid is contacted with theimaging agent precursor. In certain embodiments, the fluoride species isadsorbed onto a solid support (e.g., an anion exchange column), and asolution comprising the additive is used to elute the fluoride speciesfrom the solid support. The eluted solution is then contacted with theimaging agent precursor, or is concentrated to produce a solid, which isthen contacted with the imaging agent precursor.

In some embodiments, the additive is a bicarbonate salt. In certainembodiments, it has been discovered that substitution of a carbonatesalt with a bicarbonate salt (such as KHCO₃) results in considerableimprovement of both fluorination efficiency and starting materialintegrity. As used herein, the term “bicarbonate salt” refers to a saltcomprising a bicarbonate or hydrogen carbonate ion (HCO₃ ⁻ ion). Thebicarbonate salt may be a metal bicarbonate, such as sodium bicarbonate,calcium bicarbonate, potassium bicarbonate, magnesium bicarbonate, andthe like. In certain embodiments, the bicarbonate salt is potassiumbicarbonate (KHCO₃). In some embodiments, the bicarbonate salt comprisesa non-metal counterion, such as ammonium bicarbonate. For example, thebicarbonate salt may be a tetraalkylammonium bicarbonate salt having theformula, R₄NHCO₃, wherein R is alkyl. In some embodiments, R may be alower alkyl, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, or thelike. In certain embodiments, the ammonium salt is Et₄NHCO₃. In otherembodiments, the salt is Me₄NHCO₃, i-Pr₄NHCO₃, n-Pr₄NHCO₃, n-Bu₄NHCO₃,i-Bu₄NHCO₃, or t-Bu₄NHCO₃.

As described further in Example 14, it is thought that reactionconditions which activate larger differential rates of fluorinationwould result in a more efficient and chemoselective process; that is, adecreased rate of hydrolysis or increased rate of fluorination wouldresult. The studies outlined herein revealed that although required foranion exchange, K₂CO₃ did little to enhance fluorination over baselinelevels and served primarily a detrimental role in the fluorinationreaction. However, in contrast, addition of KHCO₃ produced a markedincrease in fluorination over the same concentration range, whiledecomposition pathways remained poorly differentiated. These facts,coupled with the observation that [¹⁸F]NaF exchange withtetraalkylammonium cations can directly produce a highly activenucleophilic fluoride source, led to investigation of a series of saltsin an effort to identify related counterion affects that increase therate of fluorination.

A comprehensive screen of ammonium salts identified a dramaticenhancement of fluorination efficiency in the presence of bicarbonateanion (e.g., see Table 1); only modest dependency on size of the alkylsubstituent was observed within the series methyl→ethyl→butyl (e.g.,Example 14).

Subsequent optimization of salt stoichiometry revealed that at levels aslow as 25 mol % of the tetraalkylammonium bicarbonate to an imagingagent precursor (e.g., 0.25:1) resulted in near complete conversion ofthe imaging agent precursor to the imaging agent; once again,unproductive consumption of starting material occurred with increasingbase concentration revealing an optimum stoichiometry range for themodified reaction conditions. Related studies directed towarddetermination of the optimal precursor concentration revealed aconcentration threshold.

This reagent combination also demonstrated rapid conversion andsignificantly improved chemoselectivity toward fluorination over theK₂CO₃/Kryptofix® 222 method. In fact, a more detailed evaluation ofcrude reaction mixtures revealed a dramatic reduction in overalldecomposition rates as evidenced by the absence of hydrolytic impurities(e.g., as described in Example 14); a result which may be attributed toa lower solution pH in the absence of Kryptofix® 222 (5-6 vs. 9-10).

In some embodiments, the additive is a salt comprising a cation thatforms a weakly coordinating salt with a fluoride species. As usedherein, a “cation that forms a weakly coordinating salt with a fluoridespecies” refers to a cation that renders a fluoride species reactivewithin a fluorination reaction. For example, the cation may not stronglybind to the fluoride species, allowing the fluoride species to act as anucleophile during a nucleophilic fluorination reaction, Those ofordinary skill the art would be able to select an appropriate cationthat would be suitable as a weakly coordinating counterion for afluoride species. For example, the cation may be have a relatively largeatomic radius and/or may be a weak Lewis base. In some cases, the cationmay be selected to be lipophilic. In some cases, the cation may compriseone or more alkyl groups. Examples of weakly coordinating cationsinclude cesium ions, ammonium ions, and the like. Examples of weaklycoordinating cations include weakly coordinating salts ofhexamethylpiperidindium, S(NMe₂)₃, P(NMe₂)₄, tetraaalkylphosphoniumsalts, tetraarylphosphonium salts, (e.g. tetraphenylphosphonium),hexakis(dimethylamino)diphosphazenium, tris(dimethylamino)sulfonium,etc.

In some embodiments, the additive is an ammonium salt, i.e., a saltcomprising a substituted or unsubstituted ammonium ion. In some cases,the ammonium ion is a weakly coordinating cation. In some cases, theammonium salt has the formula, R₄NX, where each R can be the same ordifferent and is alkyl, heteroalkyl, aryl, heteroaryl, or heterocyclic,each optionally substituted, and X is a negatively charged counterion.In some cases, R is alkyl, heteroalkyl, aryl, heteroaryl, orheterocyclic, each optionally substituted. The ammonium salt may includea wide range of negatively charged counterions, including halides,carbonates, bicarbonates, and the like. Examples of ammonium saltsinclude, but are not limited to, ammonium bicarbonate salts, ammoniumhydroxide salts, ammonium acetate salts, ammonium lactate salts,ammonium trifluoroacetate salts, ammonium methanesulfonate salts,ammonium p-toluenesulfonate salts, ammonium nitrate salts, ammoniumhalide salts (e.g., ammonium iodide salts), ammonium bisulfate salts,and the like.

In one set of embodiments, the ammonium salt is a tetraalkylammoniumsalt, such as a tetraalkylammonium bicarbonate salt. For example, theammonium salt may have the formula, R₄NHCO₃, wherein each R isindependently alkyl. In some cases, R is optionally substituted. In someembodiments, the alkyl group is a lower C₁-C₆ alkyl group. In someembodiments, the tetraalkylammonium salt is a basic tetraalkylammoniumsalt.

The salt additive (e.g., bicarbonate salt and/or ammonium salt) may beutilized in the reaction such that the molar ratio of the salt additiveto the imaging agent precursor is less than about 1.5:1. In some cases,the molar ratio is about 1.5:1 or less, about 1.4:1 or less, about 1.3:1or less, about 1.25:1 or less, about 1.2:1 or less, about 1.1:1 or less,about 1:1 or less, about 0.75:1 or less, about 0.5:1 or less, about0.25:1 or less, about 0.1:1 or less, or about 0.05:1 or less. In somecases, the ratio is greater than about 0.05:1, greater than about0.01:1, or greater than about 0.25:1. In some embodiments, the molarratio of salt additive to imaging agent precursor is about 0.5:1 toabout 1:1, or about 0.25:1 to about 1:1, or about 0.25:1 to about0.75:1, about 1.49:1 to about 0.05:1, or between about 1.4:1 to about0.25:1, or between about 0.25:1 and about 1.4:1, or between about 0.25:1and about 1.25:1.

Without wishing to be bound by theory, the use of bicarbonate andammonium salts may aid in decreasing the rate of competing reactionssuch as hydrolysis during nucleophilic fluorination of an imaging agentprecursor.

In some embodiments, the additive may be used in combination with aspecies capable of enhancing the reactivity of the fluoride species orotherwise facilitating conversion of the imaging agent precursor to theimaging agent. For example, the species may be a compound capable ofchelating one or more ions (e.g., metal ions) that may be present withinthe reaction mixture. Without wishing to be bound by theory, the speciesmay be used to chelate a counterion to a fluoride species, such as apotassium ion, thereby increasing the reactivity (e.g., nucleophilicity)of the fluoride species. In certain embodiments, the additive is used incombination with a multidentate ligand, such as a crown ether or acryptand that is capable of chelating a metal ion. The multidentateligand (e.g., cryptand) may be selected based on the metal ion to bechelated. The multidentate ligand may be, for example,4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane (e.g.,Kryptofix® 222). Other cryptands will be known to those of ordinaryskill in the art.

Some embodiments may involve the use of a bicarbonate salt incombination with4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane. In aspecific embodiment, potassium bicarbonate may be used in combinationwith 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane.

In another set of embodiments, it may be advantageous to utilize themethods described herein in the absence of a cryptand. The term“cryptand” is given its ordinary meaning in the art and refers to a bi-or a polycyclic multidentate ligand for a cation. For example, themethod may be carried out using an ammonium salt, in the absence of acryptand (e.g., such as4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane).

In another set of embodiments, the method is performed in the absence ofa carbonate salt.

In some embodiments, the use of a salt additive in the reactionincreases the yield by about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%,about 300%, about 400%, about 500%, or greater, relative to conductingthe reaction under essentially the same conditions but in the absence ofa salt additive.

Those of ordinary skill in the art will be able to select and/ordetermine the appropriate set of reaction conditions (e.g.,concentration, temperature, pressure, reaction time, solvents, etc.)suitable for use in a particular application. The imaging agent may befurther processed using one or more purification techniques, and mayoptionally be combined with additional components, such as a stabilizingagent.

Those of ordinary skill in the art would be able to select a source of afluoride species suitable for use in the methods described herein. Theterm “fluoride species” as used herein refers to a fluoride atom orgroup of atoms comprising at least one fluoride atom, wherein thefluoride atom is capable of reacting with another compound (e.g., animaging agent precursor). In some embodiments, an isotopically-enriched¹⁸F species may be produced by the nuclear reaction ¹⁸O (p,n)¹⁸F fromproton bombardment of [¹⁸O]H₂O in a cyclotron. The method may involvetreating a solution of the ¹⁸F species to remove any impurities, such asunreacted [¹⁸O]H₂O. For example, a solution of the ¹⁸F species may befiltered through an anion exchange column, where the ¹⁸F species isretained on the cationic resin matrix while the [¹⁸O]H₂O is eluted. The¹⁸F species is then removed by washing the anion exchange column withvarious mixtures of solvents and optional additives (e.g., saltadditives), forming an ¹⁸F-containing solution. In some cases, the anionexchange column is washed with an aqueous solution of a salt, such asKHCO₃ or Et₄NHCO₃.

In some cases, the ¹⁸F-containing solution is combined with additionalcomponents prior to reaction with an imaging agent precursor. Forexample, one or more solvents may be added to dilute the ¹⁸F-containingsolution to a selected concentration. In one set of embodiments, the¹⁸F-containing solution is diluted with acetonitrile.

In some cases, the ¹⁸F-containing solution may be concentrated todryness by exposure to elevated temperature and/or reduced pressure toform an anhydrous ¹⁸F-containing solid. In some embodiments, the¹⁸F-containing solid may further comprise one or more additives (e.g.,salt additives). The chemical composition of the ¹⁸F-containing solidmay depend on the number and kind of additives used in preparation ofthe ¹⁸F-containing solution. For example, a solution of potassiumbicarbonate may be used to elute the ¹⁸F species from the anion exchangecolumn, thereby resulting in an ¹⁸F-containing solid comprising [¹⁸F]KF.In another example, a solution of ammonium bicarbonate is used to elutethe ¹⁸F species from the anion exchange column, thereby resulting in an¹⁸F-containing solid comprising [¹⁸F]Et₄NF.

In some cases, the solution comprising the ¹⁸F species is heated to atemperature ranging from room temperature to about 200° C. In someembodiments, the solution is heated to a temperature ranging from90-120° C. In some cases, the solution is heated to about 75° C., about85° C., about 95° C., about 105° C., about 115° C., about 125° C., orgreater. In some cases, the solution is placed under a reduced pressureof about 100 mm Hg, about 125 mm Hg, about 150 mm Hg, about 175 mm Hg,about 200 mm Hg, about 225 mm Hg, about 250 mm Hg, about 275 mm Hg,about 300 mm Hg, about 325 mm Hg, about 350 mm Hg, about 375 mm Hg,about 400 mm Hg, or greater. In some cases, the solution is placed undera reduced pressure of about 100 mbar, about 125 mbar, about 150 mbar,about 175 mbar, about 200 mbar, about 225 mbar, about 250 mbar, about275 mbar, about 280 mbar, about 300 mbar, about 325 mbar, about 350mbar, about 375 mbar, about 400 mbar, about 450 mbar, about 500 mbar, orgreater. Those of ordinary skill in the art would be able to selectand/or determine conditions suitable for a particular reaction. In someembodiments, the solution is concentrated to dryness at about 150 mm Hgand about 115° C. In some embodiments, the solution is concentrated todryness at about 375 mm Hg and about 115° C. In some embodiments, thesolution is concentrated to dryness at about 400 mbar and about 110-150°C. In some embodiments, the solution is concentrated to dryness at about280 mbar and about 95-115° C.

The fluoride species and/or the additive, if present, is then contactedwith the imaging agent precursor under conditions that result inconversion of the imaging agent precursor to the imaging agent productvia nucleophilic fluorination. Those of ordinary skill in the art wouldbe able to select conditions suitable for use in a particular reaction.For example, the ratio of fluoride species to imaging agent precursormay be selected to be about 1:10,000 or more, about 1:5000 or more,about 1:3000 or more, about 1:2000 or more, 1:1000 or more, 1:500 ormore, 1:100 or more, 1:50 or more, 1:10 or more, 1:5 or more, or, insome cases, 1:1 or more. In some embodiments, the fluoride species maybe present at about 10 mol %, or about 5 mol %, or about 3 mol %, orabout 2 mol %, or about 1 mol % or about 0.5 mol %, or about 0.1 mol %,or about 0.05 mol %, or about 0.01 mol % relative to the amount ofimaging agent precursor. In some embodiments, at least of the fluoridespecies provided is enriched in ¹⁸F. For example, the ratio of ¹⁸Fspecies to imaging agent precursor may be selected to be about1:1,000,000 or more, or about 1:500,000 or more, or about 1:250,000 ormore, or about 1:100,000 or more, or about 1:50,000 or more, or about1:25,000 or more, or about 1:10,000 or more, about 1:5000 or more, about1:3000 or more, about 1:2000 or more, 1:1000 or more, 1:500 or more,1:100 or more, 1:50 or more, 1:10 or more, 1:5 or more, or, in somecases, 1:1 or more.

In some embodiments, the nucleophilic fluorination reaction is carriedout in the presence of one or more solvents, for example, an organicsolvents, a non-organic solvent (e.g., an aqueous solvent), or acombination thereof. In some cases, the solvent is a polar solvent or anon-polar solvent. In some embodiments, the solvent is an aqueoussolution, such as water. The solvent comprises at least about 0.001%water, at least about 0.01% water, at least about 0.1% water, at leastabout 1% water, at least about 5%, at least about 10%, at least about20% water, at least about 30% water, at least about 40% water, at leastabout 50% water, or greater. In some cases, the solvent may comprisebetween about 0.1% and 100% water, about 1% to about 90%, about 1% toabout 70%, about 1% to about 50%, or about 10% to about 50%. In somecases, the solvent comprises no more than 10% water, 5% water, 4% water,3% water, 2% water, 1% water, or 0.5% water. In some cases, the solventcomprises between about 0.01% water and about 5% water, or between about0.01% water and about 2% water, or between about 0.1% water and about0.2% water.

Other non-limiting examples of solvents useful in the inventive methodsinclude, but are not limited to, non-halogenated hydrocarbon solvents(e.g., pentane, hexane, heptanes, cyclohexane, etc.), halogenatedhydrocarbon solvents (e.g., dichloromethane, chloroform, fluorobenzene,trifluoromethylbenzene, etc.), aromatic hydrocarbon solvents (e.g.,toluene, benzene, xylene, etc.), ester solvents (e.g., ethyl acetate,etc.), ether solvents (e.g., tetrahydrofuran, dioxane, diethyl ether,dimethoxyethane, etc.), and alcohol solvents (e.g., ethanol, methanol,propanol, isopropanol, etc.). Other non-limiting examples of solventsinclude acetone, acetic acid, formic acid, dimethyl sulfoxide, dimethylformamide, acetonitrile, and pyridine. In some embodiments, the reactionis carried out in a polar solvent, such as acetonitrile.

In one set of embodiments, an anhydrous ¹⁸F-containing solid, optionallycomprising an additive, may be contacted with a solution of an imagingagent precursor (e.g., a tosylate precursor), and the resulting solutionis heated to an elevated temperature for a select period of time. Thesolution may be, for example, an acetonitrile solution. In otherembodiments, a solution of the ¹⁸F species and additive, if present, iscontacted with a solid imaging agent precursor or a solution of theimaging agent precursor.

Some embodiments involve contacting the imaging agent precursor with thefluoride species in a solution having a pH below about 7, below about 6,or, below about 5. In some cases, the solution has a pH between about 5and about 6, or between about 5 and, about 7 or between about 4 andabout 7.

In some cases, the solution comprising the ¹⁸F species, imaging agentprecursor, and, optionally, additive, is heated at an elevatedtemperature for a period of time. For example, the solution may beheated to about 50° C., about 60° C., about 70° C., about 80° C., about90° C., about 100° C., about 110° C., about 120° C., 150° C., about 170°C., about 200° C., about 225° C., about 250° C. or greater, for a periodof 5 minutes or less, 10 minutes or less, 20 minutes or less, 30 minutesor less. It should be understood that other temperatures and reactiontimes may be used. Upon completion of the reaction, the reaction mixtureis then cooled (e.g., to room temperature) and optionally diluted with asolvent, such as water.

Upon completion of the fluorination reaction, the resulting imagingagent is optionally subjected to one or more purification steps. In somecases, the synthesis, purification, and/or formulation of an imagingagent (e.g., a compound comprising formula (II)) may be prepared usingan automated reaction system comprising a cassette, wherein the cassettemay comprise a synthesis module, a purification module, and/or aformulation module. Automated reaction systems and cassettes aredescribed herein.

Purification and isolation may be performed using methods known to thoseskilled in the art, including separation techniques like chromatography,or combinations of various separation techniques known in the art, forexample, extractions, distillation, and crystallization. In oneembodiment, high performance liquid chromatography (HPLC) is used with asolvent, or mixture of solvents, as the eluent, to recover the product.In some cases, the eluent includes a mixture of water and acetonitrile,such as a 45:55 water:acetonitrile mixture. The content of water in theeluent may vary from, for example, about 1% to about 50%. In some cases,HPLC may be performed using a C18 column

The product may be further processed using additional purificationtechniques, such as filtration. In some cases, the imaging agent may bepurified using HPLC, to produce a solution of HPLC mobile phase and theimaging agent. The HPLC mobile phase may be subsequently exchanged for asolution of ascorbic acid or a salt thereof, and ethanol solution, byfiltration through a C-18 resin (e.g., C18 Sep-Pak® cartridge). In someembodiments, the solution of the HPLC mobile phase and the imaging agentis filtered through a C-18 resin, where the imaging agent remains on theresin and the other components, such as acetonitrile and/or othersolvents or components, are removed via elution. The C-18 resin may befurther washed with a solution of ascorbic acid or a salt thereof, andthe filtrate discarded. To recover the purified imaging agent, the C-18resin is washed with a solvent, such as ethanol, and the resultingsolution is optionally further diluted with an ascorbic acid solution ora salt thereof, as described herein.

Optionally, the recovered product is combined with one or morestabilizing agents, such as ascorbic acid or a salt thereof. Forexample, a solution comprising the purified imaging agent may be furtherdiluted with a solution of ascorbic acid or a salt thereof. As describedherein, a formulation may be prepared via an automated reaction systemcomprising a cassette.

In some cases, a solution comprising the imaging agent product may besterile filtered (e.g., using a 13 mm diameter, Millipore, Millex PVDF0.22 μm sterilizing filter) into a sterile product vial. The sterileproduct vial may be a commercially available, pre-sterilized unit thatis not opened during the production process, as any imaging agents (orother components) may be aseptically inserted through the septum priorto use. Those of ordinary skill in the art would be able to selectsuitable vials and production components, including commerciallyavailable, pre-sterilized units comprising a 0.22 μm pore size membraneventing filter and quality control sampling syringes.

Following aseptic filtration, individual doses may be filled insyringes, labeled, and shipped to a clinical site. Dosing administrationtechniques, kits, cassettes, method and systems (e.g., automatedreaction systems) for synthesis of the imaging agent, and testingprocedures are described herein. In some embodiments, the product isdispensed into a 3 or 5 mL syringe and labeled for distribution. Labelsmay be prepared at a radiopharmacy and applied to a syringe shield andshipping container. Additional labels may be provided in the shippingcontainer for inclusion in clinical site records.

The imaging agents may be used in a method of imaging, including methodsof imaging a patient comprising administering the imaging agent to thepatient by injection, infusion, or any other method, and imaging an areaof the patient, as described herein. In some embodiments, a portion of aheart of the patient is imaged.

Exemplary Methods for the Synthesis of Imaging Agent Precursors

Methods are also provided for synthesizing imaging agent precursors, andintermediates thereof. In some cases, the methods for synthesizing animaging agent precursor (e.g., a compound comprising formula (I))exhibits improved yields and/or may allow for the large-scale synthesisof the imaging agent precursors and/or intermediates thereof. Someembodiments provide the ability to synthesize a desired product withoutneed for purification, such as chromatography, which can betime-consuming and/or expensive with the loss of product. As notedabove, FIG. 1 shows an illustrative example of an imaging agentprecursor which has been utilized in the synthesis of an imaging agentfor imaging myocardial perfusion. The leaving group (i.e., tosylategroup) is replaced with an imaging moiety, for example, ¹⁸F, asdescribed herein, thereby forming an imaging agent.

In some embodiments, an imaging agent precursor is formed via a reactionin which a bond between a heteroatom and an alkyl, heteroalkyl, aryl, orheteroaryl group is formed. For example, the reaction may be analkylation reaction, such as an etherification reaction. In someembodiments, the reaction involves a hydroxyl-containing nucleophilicspecies reacting with an electrophilic species to form an ether linkage.As used herein, the term “ether” or “ether linkage” is given itsordinary meaning in the art and refers to the group, R^(a)—O—R^(b),where R^(a) and R^(b) can be the same or different and are alkyl,heteroalkyl, aryl, or heteroaryl, any of which may be substituted. Forexample, the reaction may involve nucleophilic addition of the oxygenatom of the hydroxyl-containing species to an electrophilic species. Insome embodiments, the reaction may involve coupling between two alcoholsvia, for example, a Mitsunobu reaction.

In some cases, the etherification reaction includes formation of a bondbetween an oxygen atom and an alkyl, aryl, heteroalkyl, heteroaryl,carbocyclic, or heterocyclic group. FIG. 3 shows an illustrativeembodiment of an etherification reaction between benzenedimethanol 12and dichloropyridazinone 11 to form the benzyl alcohol 13. In anotherembodiment, FIG. 4 shows an etherification reaction betweenhydroxychloropyridazinone 17 and methyl 4-bromomethylbenzoate to affordpyridazinone ester 18.

In some embodiments, the inventive method involves reacting a compoundcomprising formula (III):

wherein:

W is alkyl or heteroalkyl, optionally substituted;

R¹ is alkyl, optionally substituted;

R² is hydrogen or halide;

each R³ can be the same or different and is alkyl optionally substitutedwith a leaving group, or heteroalkyl optionally substituted with aleaving group; and

n is 1, 2, 3, 4, or 5;

with a nucleophile, wherein the nucleophile replaces the leaving groupto produce a product. For example, the nucleophile may be ethyleneglycol, and an etherification reaction may be carried out as describedherein. In some embodiments, the reaction is performed in the presenceof a base, such as potassium t-but oxide or potassium hydroxide. In somecases, R³ is alkyl substituted with a leaving group and/or n is 1. Insome embodiments, the compound comprising formula (III) comprises thestructure:

wherein the leaving group is Br, and the product of the reactioncomprises formula:

wherein R¹ and R² are as defined herein.

In some cases, a compound comprising formula (III) comprises thestructure:

and the product of the etherification reaction comprises the formula:

In some cases, the compound comprising formula (III) may act as anucleophile and may be reacted with an electrophile, to produce aproduct. For example, R³ may be —CH₂OH, and the electrophile may beethylene oxide.

In some embodiments, the method comprises reacting a compound comprisingformula (IV):

wherein:

R¹ is alkyl, optionally substituted;

R² is hydrogen or halide;

W is alkyl or heteroalkyl, optionally substituted;

each R⁴ can be the same or different and is alkyl optionally substitutedwith hydroxyl or heteroalkyl optionally substituted with hydroxyl; and

n is 1, 2, 3, 4, or 5;

with a reactant, wherein the hydroxyl group is replaced with a portionof the reactant to form a leaving group associated with the compound. Insome cases, R⁴ is alkyl substituted with hydroxyl and/or n is 1. In someembodiments, reacting the compound comprising formula IV involvesexposure to a halogenation reagent, such as phosphorus tribromide,pyridinium dibromide, or a combination of carbon tetrabromide andtriphenylphospine. In some embodiments, the halogenation reagent isphosphorus tribromide.

In some embodiments, W is —O(CH₂)—; R¹ is t-butyl; R² is chloride; andR⁴ is alkyl substituted with hydroxyl. In some cases, n is 1.

In some embodiments, the compound comprising formula (IV) comprises thestructure:

and the product comprises the structure:

In some embodiments, the compound comprising formula (IV) comprises thestructure:

and the product comprises the structure:

In some cases, a method is provided for synthesizing a compoundcomprising formula (IV). In some cases, the method comprisessynthesizing the compound comprising formula (IV) via an etherificationreaction between compounds comprising formulae (IVa) and (IVb):

wherein:

R¹ is alkyl, optionally substituted;

R² is hydrogen or halide;

m is 1, 2, 3, 4, or 5 or greater;

R⁵ is hydroxyl or halide; and

each R⁶ and R⁷ can be the same or different and are alkyl, heteroalkyl,or a acyl group, each optionally substituted,

wherein, when R⁵ is hydroxyl, at least one of R⁶ and R⁷ comprises aleaving group or a moiety that can be replace by a leaving group (e.g.,hydroxyl), or when R⁵ is halide, at least one of R⁶ and R⁷ comprises ahydroxyl.

In some cases, a compound comprising formula (IVa) comprises thestructure:

wherein R⁵ is as described herein.

In one set of embodiments, the compound comprising formula II issynthesized by an etherification reaction between compounds comprisingformulae (IVa) and (IVd):

wherein:

R¹ is alkyl, optionally substituted;

R² is hydrogen or halide;

R⁵ is hydroxyl or halide; and

R⁶ and R⁷ can be the same or different and are alkyl, heteroalkyl, or acarbonyl group, each optionally substituted,

wherein, when R⁵ is hydroxyl, at least one of R⁶ and R⁷ comprises aleaving group, or when R⁵ is halide, at least one of R⁶ and R⁷ comprisesa hydroxyl. In one set of embodiments, R⁵ is hydroxyl, R⁶ is a carbonylgroup, and R⁷ is a substituted alkyl. In some cases, R⁵ is hydroxyl, R⁶is an ester, and R⁷ is alkyl substituted with a leaving group.

In some cases, a compound comprising formula (IVa) comprises thestructure:

wherein R⁵ is as defined herein.

The etherification reaction may be carried out as described herein, andmay comprise exposure of the precursor compounds to a base, such aspotassium carbonate.

In some embodiments, R⁵ is halide; and R⁶ and R⁷ are each alkyl,optionally substituted. In some embodiments, R⁵ is chloride; and R⁶ andR⁷ are each alkyl substituted with hydroxyl. In one embodiment, anetherification reaction between compounds comprising formulae (IVe) and(IVf):

forms a product comprising the formula:

In one embodiment, an etherification reaction between compoundscomprising formulae:

forms a product comprising formula:

In one embodiment, an etherification reaction between compoundscomprising formulae:

forms a product comprising formula:

The product may be reduced with a reducing agent, such as lithiumaluminum hydride, lithium borohydride, or diisobutylaluminum hydride(DIBAL-H), thereby converting the ester group into an alcohol.

As shown by the illustrative embodiment in FIG. 3, benzenedimethanol 12and dichloropyridazinone 11 may be reacted via an etherificationreaction in the presence of potassium carbonate in DMF to form benzylalcohol 13. In some embodiments, a disubstituted impurity is also formedwherein benzenedimethanol 12 becomes alkylated at both hydroxyl groups,which may later be removed via chromatographic purification. Conversionof benzyl alcohol 13 to benzyl bromide 14 may be carried out with avariety of brominating agents, such as phosphorous tribromide indichloromethane. Subsequent conversion of benzyl bromide 14 to alcohol15 may be completed by reaction with ethylene glycol in the presence ofpotassium t-but oxide in tetrahydrofuran, in some cases at elevatedtemperature. Alcohol 15 may then be purified by column chromatography toremove any impurities, including disubstituted impurities formed duringthe synthesis of benzyl alcohol 13. Alcohol 15 may then be furtherreacted with p-toluenesulfonyl chloride in the presence of DMAP,triethylamine, and dichloromethane to form imaging agent precursor 1,which may be purified via recrystallization. Using the method shown inFIG. 5, the overall yield for synthesizing alcohol 15 starting fromcompound 11 (e.g., 2-(t-butyl)-4,5-dichloropyridazin-3(2H)-one) andcompound 12 (e.g., 1,4-benzenedimethanol) may be at least 10%, at least20%, at least 30%, or at least 40%, using chromatography as thepurification method. In some cases, the overall yield for synthesizingalcohol 15 starting from compound 11 and compound 12 is approximately43%, using chromatography as the purification method.

FIG. 4 shows an alternate approach to the synthesis of alcohol 13involving reaction of dichloropyridazinone 11 with potassium hydroxidein ethylene glycol to afford chlorohydroxypyridazinone 17, which maythen be reacted with methyl 4-bromomethylbenzoate in the presence ofpotassium carbonate in DMF to afford pyridazinone ester 18. Next,reduction of pyridazinone ester 18, for example, using either DIBAL-H orlithium aluminum hydride, may produce benzyl alcohol 13, which may thenbe converted to alcohol 15 and imaging agent precursor 1, as describedherein. One advantageous feature of the synthetic scheme shown in FIG. 4is the reduction or elimination of disubstituted impurities that may beformed in the synthetic scheme shown in FIG. 3. This provides theability to purify benzyl alcohol 13 without the use of chromatography.In some cases, recrystallization methods alone may be used to afford anintermediate compound of very high purity. For example, benzyl alcohol13 may be purified by recrystallization from isopropyl acetate.Additionally, the synthetic scheme shown in FIG. 4 may provide a moresimplified process, which may be performed with high-yield reactions andwithout the need for additional protection/deprotection steps. Using themethod shown in FIG. 4, the overall yield for synthesizing alcohol 15starting from compound 17 (e.g.,2-(t-butyl)-4-chloro-5-hydroxypyridazin-3(2H)-one) and methyl4-bromomethylbenzoate may be at least 10%, at least 20%, at least 30%,or at least 40%, without the use of chromatography for purification. Insome cases, the overall yield for synthesizing alcohol 15 starting fromcompound 17 and methyl 4-bromomethylbenzoate is 48%, without the use ofchromatography as a purification method.

In some embodiments, an etherification reaction (e.g., see FIG. 3,etherification reaction to form benzyl alcohol 13) is performed in thepresence of a base. The base may be, for example, a metal or a metalsalt, such as a carbonate, a metal alkoxide, or the like. In someembodiments, the base may be an organic moiety, such as an amine.Examples of bases include, but are not limited to, metals (e.g., sodiummetal), alkoxides such as sodium t-but oxide or potassium t-but oxide,an alkali metal amide such as sodium amide, lithium diisopropylamide oran alkali metal bis(trialkylsilyl)amides such as lithiumbis(trimethylsilyl)amide or sodium bis(trimethylsilyl)amide, amines(e.g., triethylamine, trimethylamine, Et(i-Pr)₂N, Cy₂MeN,4-(dimethylamino)pyridine (DMAP), 2,6-lutadine, N-methylpyrrolidine(NMP), quinuclidine), 1,5-diazabicycl[4.3.0]non-5-ene (DBN),1,5-diazabicyclo[5.4.0]undec-5-ene (DBU), ammonium salts (e.g., ammoniumhydroxide, tetramethyl ammonium hydroxide), alkali and alkaline earthcarbonates, alkali and alkaline earth bicarbonates, alkali and alkalineearth hydroxides (e.g., sodium hydroxide, potassium hydroxide), andalkali and alkaline earth hydrides, (e.g., NaH, LiH, KH, K₂CO₃, Na₂CO₃,Tl₂CO₃, Cs₂CO₃, K(Ot-Bu), Li(Ot-Bu), Na(Ot-Bu) K(OPh), Na(OPh)). In someembodiments, the base is sodium metal, sodium hydride, potassium t-butoxide, sodium methoxide, potassium carbonate, sodium carbonate, cesiumcarbonate, or potassium hydroxide. In some embodiments, the base iscesium carbonate. In some embodiments, the base is potassium hydroxide.In some embodiments, the base is sodium hydroxide. In some embodiments,the base is potassium t-but oxide. In some embodiments, the base istetramethyl ammonium hydroxide. It should be understood that anetherification reaction may also be conducted in the absence of a base.

One or more additives may be incorporated into the etherificationreaction mixture to facilitate the reaction. In some cases, theetherification reaction may be performed in the presence of a catalyst.For example, the catalyst may be a salt, such as an ammonium salt. Insome embodiments, the catalyst may be a tetraalkylammonium halide, suchas, but not limited to, tetraethylammonium iodide. In some embodiments,the catalyst may be a phase transfer catalyst. As used herein, the term“phase transfer catalyst” refers to any species capable of facilitatingthe migration of a compound from a first phase into a second, differentphase, for example, during the course of a chemical reaction. In someembodiments, the phase transfer catalyst enhances migration of acompound from one phase into a different phase, wherein a chemicalreaction takes place. Some examples of phase transfer catalysts include,but are not limited to, benzyl triethylammonium chloride,tetrabutylammonium chloride, tetraethyl ammonium chloride,tetrabutylammonium sulfate, tetraoctylammonium sulfate, and tetramethylammonium hydroxide. The phase transfer catalyst may be used incombination with, for example, a base or other chemical reagent. In someembodiments, the reaction involves exposure to sodium hydroxide and aphase transfer catalyst, such as benzyl triethylammonium chloride.

An etherification reaction may optionally be carried out in the presenceof one or more solvents. The solvent may be, for example, an organicsolvent (e.g., toluene), an aqueous solvent, or a combination thereof.In some cases, the solvent may be a polar solvent (e.g., polar proticsolvents, polar aprotic solvents). Examples of polar solvents include,but are not limited to, acetone, ethyl acetate, dimethylformamide (DMF),dimethyl sulfoxide (DMSO), acetonitrile, alcohols, or combinationsthereof. In one set of embodiments, the etherification reaction isperformed in the presence of DMF. In one set of embodiments, theetherification reaction is performed in the presence of THF. In somecases, the etherification reaction may be performed in the presence ofan ionic liquid. In some embodiments, the etherification reaction isperformed in the absence of solvent. For example, the compound may bereacted in neat ethylene glycol.

In some cases, the components of an etherification reaction is heated orcooled to any temperature from about 0° C. to about 200° C., for aperiod of time. In some embodiments, the solution is heated to atemperature from about 20° C. to about 100° C., or from about 40° C. toabout 70° C. In some cases, the solution may be heated to about 20° C.,about 30° C., about 40° C., about 50° C., about 60° C., about 70° C.,about 80° C., or greater. In some embodiments, the etherificationreaction mixture is maintained at about 20° C. In some embodiments, theetherification reaction mixture is maintained at room temperature. Insome embodiments, the etherification reaction mixture is heated to about60° C. In some embodiments, the etherification reaction mixture isheated to about 65° C. The reaction may be heated/cooled/maintained at aparticular temperature for a period of time, such as up to about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10hours, about 15 hours, about 20 hours, about 25 hours, about 30 hours,or more. In one set of embodiments, the reaction mixture is heated atabout 65° C. for about 4 hours. In another set of embodiments, thereaction mixture is maintained at about 20° C. for about 18 hours. Itshould be understood that other temperatures and reaction times may alsobe used.

In some embodiments, the method involves a reduction reaction (e.g., seeFIG. 4, reduction of pyridazinone ester 18). The term “reductionreaction” is given its ordinary meaning in the art and refers to achemical reaction in which the oxidation state of at least one atom isreduced. For example, the reduction reaction may involve reduction of anester or a ketone to an alcohol. The reduction reaction may be carriedout using reducing reagents known to those of ordinary skill in the art,including lithium aluminum hydride, lithium borohydride (with or withoutmethanol additive), and diisobutylaluminum hydride (DIBAL-H) in avariety of solvents including tetrahydrofuran, methyltetrahydrofuran,and dichloromethane. In one set of embodiments, the reduction reagentmay be a 25% w/w solution of DIBAL-H in toluene, using2-methyltetrahydrofuran as a cosolvent.

In some embodiments, the invention provides methods for the synthesis ofa compound (e.g., intermediate compound) comprising a leaving group.Leaving groups are described herein. In some embodiments, the leavinggroup is a halide, such as a bromide.

In some cases, the compound includes a moiety (e.g., hydroxyl) which maybe readily converted into a leaving group. For example, the compound mayinclude a hydroxyl group which is converted into a tosylate group uponreaction with p-toluenesulfonyl chloride. In other embodiments, acompound may include a hydroxyl group which may be treated with aphosphine (e.g., triphenylphosphine, TPP) and diethylazodicarboxylate(DEAD) using Mitsunobu chemistry to form a leaving group.

In one set of embodiments, the method involves converting a hydroxylgroup to a leaving group. For example, the method may involve replacingthe hydroxyl group with a leaving group such as a halide (e.g.,bromide). In some embodiments, the compound substituted with a hydroxylgroup is exposed to a halogenation reagent. In some cases, thehalogenation reagent is a brominating reagent, such as phosphorustribromide, pyridinium dibromide, or a combination of carbontetrabromide and triphenylphospine. In one set of embodiments, thebrominating reagent is phosphorus tribromide.

A halogenation reaction may be performed in the presence of one or moresolvents. In some embodiments, the solvent is an organic solvent, suchas dichloromethane, chloroform, benzene, or toluene. In one set ofembodiments, the solvent used is dichloromethane.

In some cases, the halogenation reaction mixture is heated or cooled toany temperature ranging from 0° C. to about 200° C., for a period oftime. In some embodiments, the solution is heated to a temperatureranging from about 20° C. to about 100° C. In some cases, the solutionis heated to about 20° C., about 30° C., about 40° C., about 50° C., orgreater, including temperatures in between. In some embodiments, thehalogenation reaction mixture is maintained at 20° C. The reaction maybe heated/cooled/maintained at a particular temperature for a period oftime, such as up to 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 10 hours, or more. In another set of embodiments, thereaction mixture is maintained at 20° C. for 30 minutes. It should beunderstood that other temperatures and reaction times may also be used.

The synthesis of an imaging agent precursor may include other reactions,including ring-opening reactions, reduction reactions,protection/deprotection reactions, and the like.

After any reaction, the compounds (e.g., intermediates, products)described herein may be subjected to one or more purification steps.Purification and isolation may be performed using methods known to thoseskilled in the art, including separation techniques like chromatography,or combinations of various separation techniques as are known the art.In some embodiments, column chromatography is used with silica oralumina as the stationary phase and a solvent, or mixture of solvents,as the eluent, to recover the product. In some cases, the eluent mayinclude a mixture of a non-polar solvent and a polar solvent. Forexample, the eluent may include a mixture of heptanes and ethyl acetate.

In some cases, the synthesis or a particular reaction may be conductedwithout need for purification. In some embodiments, a compound orintermediate may be purified using recrystallization, a process whichmay be repeated until the desired level of purity of the product isobtained. In one embodiment, the compound or intermediate isrecrystallized at least once, two times, three times, or four or moretimes to achieve the desired level of purity. For example, the compoundor intermediate may be obtained at a purity of greater than or equal to50%, 80%, 85%, 90%, 95%, 97%, 98%, 98.5%, or 99.8%. Recrystallizationmay be achieved using a single solvent, or a combination of solvents. Insome cases, recrystallization is achieved by dissolving the compound orintermediate in a solvent such as hexane at elevated temperatures, andthen cooling the solution to produce a precipitate. In certainembodiments the compound is recrystallized from hexane.

Some embodiments may involve a ring-opening reaction. For example, aring-opening reaction may be performed upon exposure of a compoundcomprising a ring to a nucleophile, optionally in the presence of acatalyst. In some embodiments, the nucleophile may be a hydride (e.g.,H). In some embodiments, the ring-opening reaction may be performed inthe presence of a metal-containing catalyst, such as zirconium chloride.

In some embodiments, the method involves reacting a compound comprisingformula (V):

wherein:

W is alkyl or heteroalkyl, optionally substituted;

R¹ is alkyl, optionally substituted; and

R² is hydrogen or halide,

with a nucleophile or a radical species to produce a compound comprisingformula (VI),

Some embodiments involve exposure of the compound comprising formula (V)to a nucleophile. In some embodiments, the nucleophile is a hydride ion(e.g., H). In some cases, reacting the compound involves contacting thecompound with diisobutylaluminum hydride (DIBAL-H).

The ring-opening reaction may also occur via a radical reaction. Forexample, the compound comprising formula (V) may be exposed to a radicalspecies, such as a hydrogen radical (e.g., H.), in order to produce thecompound comprising formula (VI). In some embodiments, the radicalspecies may be generated by a catalyst, such as SmI₂.

In some embodiments, methods are provided for synthesizing a compoundcomprising formula (VI). For example, an etherification reaction isperformed between compounds comprising formulae (Va) and (Vb):

to form a product comprising the formula:

wherein:

R¹ is alkyl, optionally substituted; and

R² is hydrogen or halide.

For example, an etherification reaction between compounds comprising theformulae:

forms a product comprising the formula:

This etherification reaction may be performed as described herein andmay involve exposure to a base (e.g., cesium carbonate, sodiumhydroxide, tetramethyl ammonium hydroxide), optionally in the presenceof a phase transfer catalyst. In some embodiments, the etherificationreaction involves exposure to sodium hydroxide and benzyltriethylammonium chloride. In some cases, the etherification reaction isperformed in the presence of a phase transfer catalyst and an ionicliquid.

In one set of embodiments, an etherification reaction between compoundscomprising formulae (Vc) and (Vb):

under Mitsunobu conditions (e.g., PPh₃ and DEAD) forms a productcomprising the formula:

wherein R¹ is alkyl, optionally substituted; and

R² is hydrogen or halide.

For example, an etherification reaction between compounds comprisingformulae:

under Mitsunobu conditions (e.g., PPh₃ and DEAD) forms a productcomprising the formula:

Some embodiments may further involve the synthesis of a compoundcomprising formula (VII):

wherein R^(a) may be hydrogen, hydroxyl, halide (e.g., chloride),O-alkyl, O-heteroalkyl, O-aryl, O-heteroaryl, S-alkyl, S-heteroalkyl,S-aryl, S-heteroaryl, alkyl, heteroalkyl, aryl, or heteroaryl, any ofwhich may be optionally substituted. In some cases, R^(a) is O-alkylsuch as O-methyl, O-ethyl, O-propyl, and the like. In some embodiments,R^(a) is O-methyl. For example, the method may involve the reaction ofmethyl 4-formyl benzoate with ethylene glycol in the presence of an acidto produce a compound comprising formula (VII). The compound comprisingformula (VII) may be further reacted, for example, to install a leavinggroup on the compound. In some cases, the leaving group is a hydroxylgroup. In one set of embodiments, R^(a) is methyl, and the carboxy groupis treated with a reducing agent such as lithium aluminum hydride,sodium bis(2-methoxyethoxy)aluminum hydride or lithium borohydride toproduce a benzylic alcohol.

FIG. 5 shows an illustrative embodiment for synthesizing alcohol 15using a ring-opening reaction. The first step involves the conversion ofether methyl 4-formyl benzoate or 4-formylbenzoic acid to thecorresponding acetal through the reaction with ethylene glycol in thepresence of an acid. In some embodiments, methyl 4-formyl benzoate andethylene glycol are reacted in the presence of toluenesulfonic acid andtoluene. The solvent may be heated at reflux, using azeotropicdistillation to remove any water that is produced in order to drive thereaction to completion. The derived acid or ester 19 may then be reducedto benzyl alcohol 20 with lithium aluminum hydride, sodiumbis(2-methoxyethoxy)aluminum hydride, lithium borohydride (e.g., for anester), or borane (e.g., for an acid). In some cases, lithium aluminumhydride or sodium bis(2-methoxyethoxy)aluminum hydride may be used asthe reducing agent. Benzyl alcohol 20 may then be reacted withdichloropyridazinone 11 via an etherification reaction as describedherein to afford compound 21. For example, the etherification reactionmay be carried out with cesium carbonate, potassium carbonate, or sodiumhydroxide in the presence of a variety of phase transfer catalysisreagents, such as, but not limited to, benzyl triethylammonium chloride.In one set of embodiments, the etherification reaction involves the useof cesium carbonate in dimethylformamide. In another set of embodiments,the etherification reaction involves the use of sodium hydroxide with1-10% benzyl triethylammonium chloride in toluene.

The acetal ring of compound 21 may then be opened to the correspondingalcohol 15 using diisobutylaluminum hydride (DIBAL-H). In some cases,the ring-opening reaction may be carried out in the presence of acatalyst, such as a metal-containing catalyst (e.g., zirconium chloride)or an organic catalyst (e.g., 9-borabicyclononane (9-BBN) dimer).

In some cases, the components of the ring-opening reaction is heated orcooled to any temperature from about −78° C. to about 200° C., for aperiod of time. In some embodiments, the reaction mixture may bemaintained at any temperature from about −78° C. to about roomtemperature. In some cases, the reaction mixture may be maintained atabout −60° C., about −50° C., about −40° C., about −30° C., about −20°C., about −10° C., about 0° C., including all temperatures in between,or greater. In some embodiments, the ring-opening reaction mixture maybe maintained at −40° C. In some embodiments, the ring-opening reactionmixture may be maintained at room temperature. The reaction may beheated/cooled/maintained at a particular temperature for a period oftime, such as about 10 minutes, about 30 minutes, about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, orany amount of time in between, or more. In another set of embodiments,the reaction mixture may be maintained at about −40° C. for about 1hour. It should be understood that other temperatures and reaction timesmay also be used.

Purification of compound 16 may be performed by successiverecrystallizations from cumene and/or isobutyl acetate. For example, seeExample 37E.

Using the method shown in FIG. 6, the overall yield for synthesizingalcohol 15 starting from methyl 4-formylbenzoate may be at least 10%, atleast 20%, at least 30%, at least 40%, or at least 50%, without or withthe use of chromatography for purification. In some cases, the overallyield for synthesizing alcohol 15 starting from methyl 4-formylbenzoateis approximately 50%, without the use of chromatography forpurification.

Any of the methods for synthesizing an imaging agent precursor describedherein may further comprise the act of exposing the compound comprisingformula (VIII):

with a reagent comprising a leaving group to form a compound comprisingformula (IX):

wherein W is alkyl or heteroalkyl, optionally substituted;

R¹ is alkyl, optionally substituted;

R² is hydrogen or halide; and

L is a leaving group.

In some cases, the reagent is a sulfonate-containing species and theleaving group is a sulfonate-containing group (e.g., asulfonate-containing precursor of an imaging agent).

In some embodiments, the sulfonate-containing group is mesylate,tosylate, or triflate. In one set of embodiments, thesulfonate-containing group is tosylate. Additional examples of leavinggroups are described herein.

For example, the act of exposing the compound comprising the formula:

to a reactant comprising a leaving group forms a product comprising theformula:

wherein R¹, R², and L are as described herein.

In one embodiment, exposure of a compound comprising the formula:

to a reactant comprising a tosylate group forms the product comprisingthe formula:

Some embodiments for synthesizing an imaging agent precursor describedherein provide novel compounds (e.g., intermediates). In someembodiments, the compound comprises the structure:

Exemplary Methods and Applications of Imaging agents

In some embodiments, the present invention relates to methods ofimaging, including methods of imaging in a subject that includesadministering a composition or formulation that includes imaging agent 1to the subject by injection, infusion, or any other known method, andimaging a region of the subject that is of interest. As describedherein,(2-t-butyl-4-chloro-5-[4-(2-(¹⁸F)fluoroethoxymethyl)-benzyloxy]-2H-pyridazin-3-1,or imaging agent 1, comprises the formula:

Imaging agent 1 binds to the mitochondrial complex I of the electrontransport chain with high affinity. Imaging agent 1 shows selectiveuptake to the heart due to the high density of mitochondria in themyocardium. Regions of interest may include, but are not limited to, theheart, cardiovascular system, cardiac vessels, blood vessels (e.g.,arteries, veins) brain, and other organs. A parameter of interest, suchas blood flow, cardiac wall motion, etc., can be imaged and detectedusing methods and/or systems of the invention. In some aspects of theinvention, methods for evaluating perfusion, including myocardialperfusion, are provided.

In some embodiments, methods of the present invention include (a)administering to a subject a composition that includes imaging agent 1,and (b) acquiring at least one image of at least a portion of thesubject. In some cases, acquiring employs positron emission tomography(PET) for visualizing the distribution of imaging agent 1 within atleast a portion of the subject. As will be understood by those ofordinary skill in the art, imaging using methods of the invention mayinclude full body imaging of a subject, or imaging of a specific bodyregion or tissue of the subject that is of interest. For example, if asubject is known to have, or is suspected of having myocardial ischemia,methods of the invention may be used to image the heart of the subject.In some embodiments, imaging may be limited to the heart, or may includethe heart and its associated vascular system.

In some embodiments of the invention, methods of diagnosing or assistingin diagnosing a disease or condition, assessing efficacy of treatment ofa disease or condition, or imaging in a subject with a known orsuspected cardiovascular disease or condition are provided. Acardiovascular disease can be any disease of the heart or other organ ortissue nourished by the vascular system. The vascular system includescoronary arteries, and all peripheral arteries supplying nourishment tothe peripheral vascular system and the brain, as well as veins,arterioles, venules, and capillaries. Examples of cardiovasculardiseases include diseases of the heart, such as coronary artery disease,myocardial infarction, myocardial ischemia, angina pectoris, congestiveheart failure, cardiomyopathy (congenital or acquired), arrhythmia, orvalvular heart disease. In some embodiments, the methods disclosedherein are useful for monitoring and measuring coronary artery diseaseand/or myocardial perfusion. For example, a method described herein candetermine the presence or absence of coronary artery disease and/or thepresence or absence of myocardial infarct. Conditions of the heart mayinclude damage, not brought on by disease but resulting frominjury—e.g., traumatic injury, surgical injury. In some cases, methodsof the invention may include determining a parameter of, or the presenceor absence of, myocardial ischemia, rest (R) and/or stress (S)myocardial blood flows (MBFs), coronary flow reserve (CFR), coronaryartery disease (CAD), left ventricular ejection fraction (LVEF),end-systolic volume (ESV), end-diastolic volume (EDV), and the like.

In some cases, a subject to whom a method of the invention is applied,may have signs or symptoms suggestive of myocardial ischemia ormyocardial infarction. In some cases methods of the invention can beused to identify early or pre-disease conditions that indicate that asubject is at increased risk of a disease. In some instances, methods ofthe invention can be used to determine a subject's risk of futurecardiac events such as myocardial infarction or cardiac death. Imagingmethods of the invention can be used to detect myocardial ischemia insubjects already diagnosed as having a myocardial ischemia disorder orcondition, or in subjects that have no history or diagnosis of such acondition. In other instances, methods of the invention can be used toobtain measurements that provide a diagnosis or aid in providing adiagnosis of a myocardial ischemia disorder or condition. In someinstances, a subject may be already be undergoing drug therapy for amyocardial ischemia disorder or condition, while in other instances asubject may not be undergoing therapy for myocardial ischemia. In someembodiments, methods of the invention can be used to assess efficacy ofa treatment for a disease or condition. For example, the heart can bevisualized using imaging agents of the invention before, during, and/orafter treatment of a condition affecting the heart of a subject. Suchvisualization may be used to assess a disease or condition, and aid inselection of a treatment regimen, e.g. therapy, surgery, medications,for the subject.

A PET imaging agent may have a high first-pass extraction fraction andcan track regional myocardial blood flow over a wide range. Thesefeatures may permit detection of milder decreases in coronary flowreserve and accurate estimation of absolute myocardial blood flow (MBF).PET imaging agents of the invention provide these and other features andare also available as a unit dose from regional PET radiopharmacies,obviating the need for on-site cyclotrons or costly Rb-82 generators.

In some embodiments of the invention, imaging agent 1 is used as animaging agent in combination with positron emission tomography (PET) orwith other imaging methods including, but not limited to SPECT imaging.In some embodiments of the invention, imaging agent 1 is administered toa subject and imaged in the subject using PET. As will be known to thoseof ordinary skill in the art, PET is a non-invasive technique thatallows serial images and measurements to be obtained in a single subjectover a time period. PET imaging used in methods of the invention may becarried out using known systems, methods, and/or devices. In someembodiments of the invention, PET imaging is conducted using a cardiacimaging system. A cardiac imaging system may include PET imagingfunctionality and a control unit configured to drive the imagingfunctionality to perform a PET imaging procedure on a portion of thesubject before, during, and/or after administration of imaging agent 1to the subject. In some cases, the control unit is configured to drivethe imaging functionality to perform a PET imaging procedure. Thecontrol unit may comprise a computer system and/or software. In such acase, the computer system may be programmed or configured to execute therequired methods for acquiring and/or analyzing the images. Further, thesystem may include a data storage device that is readable by a machine,embodying a set of instructions executable by the machine to perform therequired methods of acquiring and/or analyzing the images.

The useful dosage of the imaging agent to be administered and theparticular mode of administration will vary depending upon such factorsas age, weight, and particular region to be imaged, as well as theparticular imaging agent used, the diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, microsphere,liposome, or the like, as described herein, and as will be readilyapparent to those skilled in the art.

In some embodiments, an imaging agent is administered at a low dosageand the dosage increased until the desirable diagnostic effect isachieved. In one embodiment, the above-described imaging agents may beadministered by intravenous injection, usually in saline solution, at adose of about 0.1 to about 100 mCi per 70 kg body weight (and allcombinations and subcombinations of dosage ranges and specific dosagestherein), or between about 0.5 and about 50 mCi, or between about 0.1mCi and about 30 mCi, or between 0.5 mCi and about 20 mCi. For use asnuclear medicine imaging agents, the imaging agents, dosages,administered by intravenous injection, may range from about 0.1 pmol/kgto about 1000 pmol/kg (and all combinations and subcombinations ofdosage ranges and specific dosages therein), and in some embodiments,less than 150 pmol/kg.

Imaging systems and components thereof will be known to those ofordinary skill in the art. Many imaging systems and components (e.g.,cameras, software for analyzing the images, etc.) are known andcommercially available, for example, a Siemens Biograph-64 scanner. Anytechnique, software, or equipment that reduces or eliminates motion instatic perfusion images may be used in methods of the invention, becausespatial blurring and artifacts can be caused by patient motion duringimage acquisition. In some embodiments of the invention, images may beacquired in list mode, and may be static, dynamic, or gated images. Anappropriate period of time for acquiring images can be determined by oneof ordinary skill in the art, and may vary depending on the cardiacimaging system, the imaging agent (e.g., amount administered,composition of the imaging agent, subject parameters, area of interest).As used herein a “period of acquiring images” or an “image acquisitionperiod” may be a period of obtaining a single continuous image, or maybe a period during which one or more individual discrete images areobtained. Thus, a period of image acquisition can be a period duringwhich one or more images of one or more regions of a subject areacquired.

In some embodiments of the invention, a period of image acquisitionafter administration of an imaging agent of the invention to a subjectmay be between about 30 seconds and about 60 minutes, between about 1minute and about 30 minutes, between about 5 minutes and about 20minutes, or at least about 1 minute, about 3 minutes, about 5 minutes,about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes,about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes,4 about 5 minutes, about 60 minutes, or greater. For example, in arest/stress imaging protocol there would be at least two periods ofimage acquisition with at least one corresponding to the rest segmentand least one corresponding to the stress segment. In some embodiments,imaging may be continuous over the imaging period of time, or images maybe acquired at intervals such as in periodic or gated imaging.

In some aspects of the invention, gated acquisition is used to acquireimages from a subject to whom an imaging agent prepared by methods ofsuch as imaging agent 1 has been administered. Gated imaging can be usedin various aspects of the invention, and for example, may provide imagesof a beating heart of a subject and may be used to attain a functionalevaluation of how well a heart is beating. Gated imaging can beperformed by acquiring separate images from the subject at specificintervals during a period of image acquisition. A non-limiting exampleof gated imaging is a case when a period of image acquisition is about10 minutes long, and images are acquired at repeated intervals duringthe 10 minute period. The frequency of acquisition of images during theperiod can be set by the operator, for example, the frequency can be atleast every about 1 msec, about 5 msec, about 10 msec, about 20 msec,about 50 msec, about 100 msec, about 125 msec, about 250 msec, or more.The length of the interval is set by the operator to be triggered by anevent, such as a cardiac R wave, with the length of the interval isdefined by the number of time bins desired per R wave to R waveinterval. Those of skill in the art will be familiar with the conceptand methods of gated image acquisition and can use known methods toobtain gated images using imaging agent 1 as an imaging agent.

Image acquisition in gated imaging can be triggered at specificintervals, for example, image acquisition can be triggered using an EKGof the heart. In a non-limiting example, an R wave-gated scanner maytrigger acquisition of an image and the mean length of time between oneR wave of a heart and the next can be stored. The number of images tocollect can then be determined. For example, a first image can beacquired at 125 msec, a second image can be acquired at 250 msec, athird image can be acquired at 375 msec, etc.—thus images in that Rinterval may be acquired at 125 msec intervals. When the next R intervalbegins, the collection of images resets and image data is then acquiredinto the “first” image at 125 msec from that R interval start time, andthen into the “second” image collected 250 msec from that R intervalstart time, etc. Thus, within each R interval image acquisition is addedinto the initial image of the series and incremented into successiveimages in the series so that a sequence of images can be collected at adesired frequency with the zero time being reset at the start of each Rinterval. Acquired gated images can be used to provide an image of heartmotion and can provide information on heart wall thickness, whether ornot one or more sections of the heart are not moving or beating (e.g. awall motion defect). Use of gated imaging may provide data with which tojudge perfusion of the heart, such as ejection fraction, and tovisualize and identify reduced, absent, paradoxical or asynchronous wallmotion. Use of gated imaging may also provide data with which to improveassessment of myocardial perfusion, judge cardiac function and tovisualize and identify asynchronous wall motion.

In some cases, PET imaging may be used to assess myocardial viabilityvia the ability of this technique to demonstrate metabolic consequencesof myocardial ischemia. Using PET imaging, myocardial segments that arelikely to improve after revascularization can be identified. In somecases, PET imaging may be used in the detection of coronary arterydisease and can also serve as an alternative test for subjects whocannot undergo treadmill exercise stress testing. In some embodiments, astress test method (e.g., pharmacological stress, exercise stress) maybe employed with PET using methods of the invention to qualitatively orquantitatively assess one or more parameters of cardiac function duringinfusion of the imaging agent. Agents for, and methods of, inducingstress, for example, using exercise or pharmacological stress are wellknown in the art. Suitable induction of stress can be carried out usingestablished, known agents and methods. Functions usefully measured usingmethods of the invention include, but are not limited to, in variousembodiments, imaging of myocardial perfusion, imaging, or measurement ofventricular function, and measuring coronary blood flow velocity.

In some cases, methods for imaging the heart of a subject may includeadministering a first dose of imaging agent 1 to the subject while thesubject is at rest, acquiring at least one first image of the heart,followed by subjecting the subject to stress (e.g., exercise stress orpharmacological stress) and administering a second dose of imaging agent1 to the subject during the period of stress, and acquiring at least oneother image of the heart.

In some embodiments, the dose of imaging agent 1 to be used duringexercise-induced stress in a rest/stress protocol is greater than thatnecessary for pharmacologically-induced stress with the ratio ofexercise-induced stress dose to pharmacologically-induced stress dosebeing greater then or equal to about 1.2, about 1.3, about 1.4, about1.5, about 1.6, about 1.7, about 1.8, about 1.9, or greater. Withrespect to pharmacological stress, in some embodiments of the inventionthat involve rest/stress imaging methods, the dose of imaging agent 1administered for imaging during the pharmacological stress is a minimumof two times the dose of imaging agent 1 administered for imaging atrest. With respect to exercise stress, in some embodiments of theinvention that involve rest/stress imaging methods, the dose of imagingagent 1 administered for imaging during the exercise-induced stress is aminimum of three times the dose of imaging agent 1 administered forimaging at rest. In some embodiments of the invention, for imaging firstat rest followed by imaging with stress, the dose of imaging agent 1administered at rest will be lower than the dose of imaging agent 1administered at stress.

In some cases, imaging methods of the invention may be completed in asingle day (e.g., less than about 24 hours, less than about 12 hours,less than about 6 hours, less than about 4 hours, less than about 2hours, less than about 1 hour), as described herein. In other cases,methods may be completed in longer periods of time, e.g. over more thanabout 24 hours, about 36 hours, or about 48 hours.

Imaging agent 1 may be provided in any suitable form, for example, in apharmaceutically acceptable form. In some cases, imaging agent 1 isincluded in a pharmaceutically acceptable composition. In someembodiments, imaging agent 1 is provided as a composition comprisingethanol, sodium ascorbate, and water. In some cases, the compositioncomprises less than 20 weight % ethanol, less than 15 weight % ethanol,less than 10 weight % ethanol, less than 8 weight % ethanol, less than 6weight % ethanol, less than 5 weight % ethanol, less than 4 weight %ethanol, less than 3 weight % ethanol, or less ethanol. In some cases,the composition comprises less than 100 mg/mL, less than 75 mg/mL, lessthan 60 mg/mL, less than 50 mg/mL, less than 40 mg/mL, less than 30mg/mL, or less sodium ascorbate in water. In a particular non-limitingembodiment, imaging agent 1 is provided as a solution in watercomprising less than 4% ethanol and less than 50 mg/mL sodium ascorbatein water.

An imaging agent 1 composition for injection may be prepared in aninjection syringe. Imaging agent 1 may be prepared by a radiopharmacy(e.g., using the methods described herein) and/or a PET manufacturingcenter and provided to a health-care professional for administration. Insome aspects of the invention, imaging agent 1 is provided, for example,in a syringe or other container, with <50 mg/mL sodium ascorbate inwater, <4 wt % ethanol, and about 1 to 14 mCi of imaging agent 1. Theamount of imaging agent 1 may vary depending on whether a rest or stressdose is being administered. For example, a higher amount of imagingagent 1 may be provided in a syringe or container for use in a stressdose administration than provided in a syringe for use in a restadministration. A dose of imaging agent 1 may be diluted with saline(e.g., as described herein), if needed to obtain a practical dosevolume. For example, if the activity concentration of imaging agent 1 isso high that only 0.1 mL is need for an appropriate dose for a subject,the solution can be diluted, e.g., with sterile saline, so the syringecontains 0.5 ml to 4 or more ml of an imaging agent 1 solution foradministration. In some embodiments of the invention, an injectionvolume for imaging agent 1 is between 0.5 and 5 ml, 1 and 4 ml, 2 and 3ml, at least 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9ml, 10 ml, or more. Those of skill in the art will recognize how todilute imaging agent 1 to produce a sufficient dose volume foradministration. In some aspects of the invention, imaging agent 1 isprovided in a container such as a vial, bottle, or syringe, and may betransferred, as necessary, into a suitable container, such as a syringefor administration.

Syringes that include an adsorbent plunger tip may result in 10 to 25%of imaging agent 1 activity remaining in the syringe after injection.Syringes lacking an adsorbent plunger tip may be used, such as a 3 or 5mL NORM-JECT (Henke Sass Wolf, Dudley, Mass.) or other equivalentsyringe lacking an adsorbent plunger tip. Reduction of adsorption in thesyringe can increase the amount of imaging agent 1 that is transferredfrom the syringe and administered to the subject in methods of theinvention. A syringe used in methods of the invention may compriseimaging agent 1, and be a non-adsorbing, or reduced adsorbent syringe.In some embodiments a non-adsorbent or reduced-adsorbent syringe is asyringe that has been coated or treated to reduce imaging agent 1adsorption. In some embodiments, a non-adsorbent or reduced-adsorbentsyringe is a syringe that lacks an adsorbent plunger tip. In someembodiments, a syringe used in conjunction with the invention adsorbsless than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of imaging agent 1 it contains. Incertain aspects of the invention, a syringe that contains imaging agent1 does not include a rubber or latex tip on the plunger. In some cases asyringe used in methods of the invention, includes a plunger thatadsorbs less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of imaging agent 1 that thesyringe contains. A syringe of the invention may also comprise sodiumascorbate, ethanol, and water, and certain embodiments of the inventioninclude a syringe containing imaging agent 1 in a solution comprisingless than 4% ethanol and less than 50 mg/mL sodium ascorbate in water. Asyringe of the invention may be a syringe that is latex free, rubberfree, and/or lubricant free. A syringe of the invention may containimaging agent 1 in an amount between about 1.5 and about 14 mCi. Asyringe of the invention may contain about 20 mCi or less of imagingagent 1.

Components of a composition comprising imaging agent 1 may be selecteddepending on the mode of administration to the subject. Various modes ofadministration that effectively deliver imaging agents of the inventionto a desired tissue, cell, organ, or bodily fluid will be known to oneof ordinary skill in the art. In some embodiments, the imaging agent isadministered intravenously (e.g., intravenous bolus injection) usingmethods known to those of ordinary skill in the art. As used herein, adose that is “administered to a subject” means an amount of the imagingagent, e.g. imaging agent 1, that enters the body of the subject. Insome embodiments, due to factors such as partial retention of imagingagent such as imaging agent 1 in a syringe, tubing, needles, catheter,or other equipment used to administer the imaging agent to a subject,the amount of an imaging agent such as imaging agent 1 that is measuredor determined to be in the a syringe or other equipment prepared foradministration may be more than the amount in the dose that isadministered to the subject. In some embodiments, an injection of animaging agent is followed by a flushing injection of normal saline, intothe subject, using the same tubing, needle, port, etc., used foradministration of imaging agent 1. Flushing may be performed immediatelyfollowing administration of imaging agent 1, or up to 1 min, 2 min, 3min, 5 min, or more, after the administration. The volume of saline orother agent for flushing may be up to 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10ml, 15 ml, 20 ml, or more. As will be understood by those of ordinaryskill in the art, in embodiments where imaging agent 1 is administeredusing a syringe or other container, the true amount of imaging agent 1administered to the subject may be corrected for any imaging agent 1that remains in the container. For example, the amount of radioactivityremaining in the container, and tubing and needle or delivery instrumentthat carried the imaging agent from the container and into the subjectcan be determined after the imaging agent has been administered to thesubject and the difference between the starting amount of radioactivityand the amount remaining after administration indicates the amount thatwas delivered into the subject. In some cases, the container orinjection device (e.g., catheter, syringe) may be rinsed with a solution(e.g., saline solution) following administration of imaging agent 1.

In some embodiments of the invention, the total amount of imaging agent1 administered to a subject over a given period of time, e.g., in onesession, is less than or equal to about 50 mCi, less than or equal to 40mCi, less than or equal to 30 mCi, less than or equal to 20 mCi, lessthan or equal to 18 mCi, less than or equal to 16 mCi, less than orequal to 15 mCi, less than or equal to 14 mCi, less than or equal to 13mCi, less than or equal to 12 mCi, less than or equal to 10 mCi, lessthan or equal to 8 mCi, less than or equal to 6 mCi, less than or equalto 4 mCi, less than or equal to 2 mCi, less than or equal to 1 mCi, lessthan or equal to 0.5 mCi. The total amount administered may bedetermined based on a single dose or multiple doses administered to asubject within a given time period of up to 1 minute, 10 minutes, 30minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, ormore.

Based on radiation dose studies, the desirable maximum dose administeredto a subject may be based on determining the amount of imaging agent 1which limits the radiation dose to about 5 rem to the critical organand/or about 1 rem effective dose (ED) or lower, as will be understoodby those of ordinary skill in the art. In a particular embodiment, thedesirable maximum dose or total amount of imaging agent 1 administeredis less than or equal to about 25 mCi, or less than or equal to about 14mCi over a period of time of up to 30 min, 1 hour, 2 hours, 6 hours, 12hours, 24 hours, 48 hours, or more. In some embodiments, the maximumdose of imaging agent 1 administered to a subject may be less than 3.5μg per 50 kg of body weight per day. That is, in some embodiments of theinvention, the maximum dose of imaging agent 1 administered to a subjectmay be less than about 0.07 μg of imaging agent 1 per kg of body weightper day.

In some embodiments, methods of the invention include administering to asubject a first dose (e.g., rest dose) of imaging agent 1 while thesubject is at rest, and performing a first PET imaging procedure (e.g.,a PET rest imaging procedure) and acquiring at least a first image of aportion of a subject. In some cases, after administering an imagingagent such as imaging agent 1 while the subject is at rest, the subjectmay be subjected to stress and during the stress a second dose (e.g.,stress dose) of an imaging agent such as imaging agent 1 is administeredto the subject, and a second PET imaging procedure (e.g., a PET stressimaging procedure) is performed on the subject and at least one otherimage of a portion of the subject may be acquired. The above is anexample of a method that may be referred to as a rest-stress test. Thetime between the completion of the first PET imaging procedure andadministration of the second imaging agent dose is referred to as thewait time. In some cases, a rest-stress test may be completed in aperiod of time of less than 48 hours, less than 36 hours, less than 24hours, less than 12 hours, less than 6 hours, less than 5 hours, lessthan 4 hours, less than 3 hours, less than 2 hours, less than 1 hour,less than 30 minutes, or less.

In some embodiments, the amount of imaging agent 1 administered in afirst dose to a subject at rest (e.g., rest dose in a rest-stress test)is between about 1 mCi and about 5 mCi, between about 2 mCi and about 4mCi, between about 2.5 mCi and about 3.5 mCi, or about 3 mCi. Followingadministration of the first dose of imaging agent 1, a PET imagingprocedure may be performed and at least one first image may be acquiredof at least a portion of the subject.

In some cases, the amount of imaging agent 1 administered to a subjectduring stress may be based on the amount of imaging agent 1 administeredto the subject at rest. That is, the dosing during stress may be based,at least in part, on a dosing ratio (DR) (e.g., ratio of stress-dose torest-dose). The DR may depend on numerous factors as will be known tothose of ordinary skill in the art, and in some cases, may depend on themethod of inducing stress in the subject. In some cases, the DR isbetween 1 and 5, between 1 and 4, between 1 and 3, between 2 and 5, orbetween 2 and 4. In some cases, the DR is at least 1, at least 1.5, atleast 2, at least 3, at least 4, or at least 5. In some cases, the DR isbetween 2.5 and 5.0, or 2.5 and 4.0, or 3.0 and 4.0, or 3.0 and 5.0times greater than the first dose of the imaging agent. In some cases,the DR required for a subject subjected to exercise stress is more thanthe DR and/or time interval used for a subject subjected topharmacological stress. This may be due, in part, to a lower netmyocardial uptake of radioactivity with exercise. In some cases, the DRemployed for a subject subjected to exercise stress is between 2 and 4,between 2.5 and 3.5, or at least 3.0, at least 3.5, at least 4.0, ormore, in embodiments wherein the wait time is at least 15 minutes 30minutes, 1 hour, 1.5 hours, 2 hours, or the like. In some cases, the DRemployed for a subject subjected to pharmacological stress is between 1and 3, or between 1.5 and 2.5, or at least 2.0, at least 2.2, or atleast 2.5, or more, in embodiments wherein the wait time is at least 15minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, or the like. In aparticular embodiment, for a subject subjected to pharmacologicalstress, a DR of at least 2.2 for a wait time of at least 15 minutes orat least 30 minutes is employed, and/or for a subject subjected toexercise stress, a DR of at least 3.0 for a wait time of at least 30minutes or at least 1 hour is employed.

In some cases, the imaging agent is between about 2.0 mCi and about 3.5mCi, or 2.4 mCi to about 2.9 mCi, or between about 2.5 mCi to about 3.0mCi, or between about 2.5 mCi and about 3.5 mCi.

In a particular embodiment, for pharmacological stress (e.g.,vasodilator stress induced by administration of adenosine orregadenoson), a dose of about 2.9 mCi to about 3.4 mCi rest is providedduring rest, and a dose of about 2.0 to about 2.4 times the rest dose isprovided during stress, with a wait time of at least about 15 minutes orat least about 30 minutes.

In some cases, the second dose of the imaging agent is between about 5.7mCi and about 6.2 mCi, or between about 6.0 mCi and about 6.5 mCi, andabout 5.7 mCi and about 6.5 mCi.

In another embodiment, for exercise stress, a dose of about 1.7 mCi toabout 2.0 mCi is provided during rest, and a dose of about 3.0 to about3.6 times the rest dose is provided during stress, with a wait time ofat least about 30 minute or at least about 60 minutes. In some cases,the second dose of the imaging agent is between about 8.6 mCi and about9.0 mCi, or between about 9.0 and about 9.5 mCi, or between about 8.6mCi and about 9.5 mCi.

In another embodiment, for pharmacological stress, a dose of betweenabout 2.4 mCi and about 2.9 mCi is administered during rest, and a dosebetween about 5.7 mCi and about 6.2 mCi is administered during stress(e.g., DR of at least about 2), wherein the wait time is at least about15 minutes or at least about 30 minutes. In another embodiment, forexercise stress, a dose of between about 1.7 mCi and about 2.0 mCi isadministered during rest, and a dose of between about 8.6 mCi and about9.0 mCi is administered during stress (e.g., DR at least about 3),wherein the wait time is at least 30 minutes or at least 60 minutes.

In a particular embodiment, for pharmacological stress, a dose of about2.9 mCi to about 3.3 mCi rest is provided during rest, and a dose of 2.0to 2.4 times the rest dose is provided during stress, with a wait timeof at least 15 minutes or at least 30 minutes. In another embodiment,for exercise stress, a dose of about 2.9 mCi to about 3.3 mCi isprovided during rest, and a dose of 3.0 to 3.6 times the rest dose isprovided during stress, with a wait time of at least 30 minute or atleast 60 minutes.

In yet another embodiment, for pharmacological stress, a dose of about2.5 mCi to about 3.0 mCi rest is provided during rest and a dose about 6mCi to about 6.5 mCi is provided during stress. In still yet anotherembodiment, for exercise stress, a dose of about 2.5 mCi to about 3.0mCi rest is provided during rest and a dose about 9 mCi to about 9.5 mCiis provided during stress.

In some embodiments, administering during the stress includes beginningadministering the second dose within a period of time after completingthe rest imaging procedure (e.g., the wait period). In some cases, thesecond dose may be administered at a period of time of at least 5minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 45minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, or greater, aftercompleting the rest imaging procedure. In some cases, the second dose isadministered at a time period of between 5 minutes and 30 days, between5 minutes and 20 days, between 5 minutes and 10 days, between 5 minutesand 5 days, between 5 minutes and 4 days, between 5 minutes and 3 days,between 5 minutes and 48 hours, between 5 minutes and 24 hours, between5 minutes and 12 hours, between 5 minutes and 2 hours, between 5 minutesand 90 minutes, between 10 minutes and 60 minutes after completing therest imaging procedure.

For stress testing in methods of the invention, a subject may besubjected to stress using procedures known to those of ordinary skill inthe art. In some cases, the subject may be subjected to stress usingprocedures including exercise stress and/or pharmacological stress.Pharmacological stress may be induced by administering to the subject apharmacological agent such as a vasodilator. Examples of usefulpharmacological stress agents, include, but are not limited toadenosine, dobutamine, dipyridamole, regadenoson, binodeneson,apadeneson, and other adenosine A2a receptor agonists. Dosing andadministration of pharmacological stress inducing agents, such asvasodilators, are well known in the art and can be determined for use inconjunction with methods and systems of the invention. Exercise stressmay be induced using a treadmill, exercise bicycle, hand crank, or otherequipment suitable to increase a subject's heart rate through increasedexertion.

In some embodiments of the invention a rest/stress method is followed.In a rest/stress method a period of rest and imaging is followed by aperiod of stress and imaging, with the order being rest first, followedby stress. In certain embodiments of the invention, a stress/rest methodmay be used. In a stress/rest method, a period of stress and imaging isfollowed by a period of rest and imaging, with the order being stressfirst, followed by rest. In some aspects of the invention, imaging agent1 can be used in a “stress only” method, in which stress is induced in asubject for imaging with imaging agent 1 with no rest imaging during thesubject session. In some embodiments of the invention, imaging agent 1can be used in a “rest only” method, in which a subject does not undergostress induction, but is only imaged with imaging agent 1 at rest inthat session.

Exemplary Cassettes and Reaction Systems

In some embodiments, systems, methods, kits, and cassettes are providedfor the synthesis of an imaging agent (e.g., imaging agent 1). In someembodiments, an imaging agent may be prepared using an automatedreaction system comprising a disposable or single use cassette. Thecassette may comprise all the non-radioactive reagents, solvents,tubing, valves, reaction vessels, and other apparatus and/or componentsnecessary to carry out the preparation of a given batch of imagingagent. The cassette allows the reaction system to have the flexibilityto make a variety of different imaging agents with minimal risk ofcross-contamination, by simply changing the cassette. By the term“cassette” is meant a piece of apparatus designed to fit removably andinterchangeably onto automated reaction systems, in such a way thatmechanical movement of moving parts of the automated reaction systemcontrols the operation of the cassette from outside the cassette, ie.,externally. In certain embodiments, a cassette comprises a lineararrangement of valves, each linked to a port where various reagents,cartridges, syringes, and/or vials can be attached, by either needlepuncture of a septum-sealed vial, or by gas-tight, marrying joints. Eachvalve may have a male-female joint which interfaces with a correspondingmoving arm of the automated synthesizer. External rotation of the armcan control the opening or closing of the valve when the cassette isattached to the automated reaction system. Additional moving parts ofthe automated reaction system are designed to clip onto syringe plungertips, and thus raise or depress syringe barrels. An automated reactionsystem may further include a controller and one or more controllablevalves in electrical communication with the controller. An automatedreaction system may also include additional vessels, valves, sensors,heaters, pressurizing elements, etc., in electrical communication withthe controller. An automated reaction system may be operated by acontroller using suitable software for control of valve openings andclosings, heating, cooling, pressure levels, fluid movement, flow rate,etc. The automated reaction system may optionally include a computeroperating system, software, controls, etc., or other component. Inaddition, the automated reaction system may comprise a mount for thecassette.

Examples of automated reaction systems (e.g., a nucleophilic reactionsystem), include, but are not limited to, the Explora GN or RN synthesissystem (Siemens Medical Solutions USA, Inc.), GE-Tracerlab-MX synthesissystem (GE Healthcare), Eckert & Zeigler Modular-Lab Synthesis system,etc., which are commonly available at PET manufacturing facilities.

The automated reaction systems may carry-out numerous steps, as outlinedin FIG. 6, including, but not limited to, preparation of the ¹⁸Ffluoride species, providing an imaging agent precursor, optionally in asolution (e.g., as described herein, for example, imaging agentprecursor 1 in acetonitrile), a radiolabeling reaction (e.g., reactionof the ¹⁸F species and the imaging agent precursor to form the imagingagent) optionally in a synthesis module, purification (e.g., bypreparative HPLC), solvent exchange (e.g., by SepPak), asepticfiltration, and release into a container. For example, see Examples 9,10, and 11.

In some embodiments, the automated reaction system may make use of acassette comprising a reaction module in fluid connection with apurification module and/or a formulation module. FIGS. 7 and 8 showschematic representations of cassettes in connection with exemplaryreaction systems for synthesizing an imaging agent comprising a reactionmodule, a purification module, and/or a formulation module.

For example, the reaction module may include a reaction chamber in whichconversion of the imaging agent precursor to the imaging agent isperformed. The reaction module may include a source of a fluoridespecies (e.g., ¹⁸F), a source of the imaging agent precursor, a sourceof an additive (e.g., salt additive), and other sources of additionalcomponents such as solvents, each of which may optionally be fluidlyconnected to the reaction chamber. The reaction module may also comprisean anion exchange column for purification of the fluoride species, priorto introduction into the reaction chamber.

Upon reaction, the resulting imaging agent product is transferred fromthe reaction module to the purification module for further processing,treatment, and/or purification. The purification module may include, forexample, a column (e.g., an HPLC column) fluidly connected to one ormore sources of solvents to be used as eluents. The purification modulemay further comprise a source of a stabilizing agent (e.g., ascorbicacid or a salt thereof), which may be added to the imaging agent uponpurification (e.g., by HPLC). The purified imaging agent is thentransferred to the formulation module, where further purification andformulation may be performed. The formulation module may include afilter for aseptic filtration and/or a C-18 column for solvent exchange.

In another embodiment, a cassette comprises a reaction module and aformulation module. A reaction module of the invention may include asource of ¹⁸F, a filter to remove unreacted [¹⁸O]H₂O, a source of anammonium salt, a source for a diluent for the ¹⁸F, a source for animaging agent precursor, (e.g., imaging agent precursor 1 shown in FIG.1, or other imaging agent precursor), a source for an H₂O diluent forthe imaging agent precursor, a reaction vessel for reacting the ¹⁸F andthe imaging agent precursor, a solid phase extraction column (e.g., aC18 column, or other suitable column) in fluid communication with thereaction vessel. The solid phase extraction column includes a solidsorbent to adsorb the radiolabeled imaging agent product on the sorbent.At least a portion of the residual reaction impurities pass throughsolid phase extraction column without adsorbing on the sorbent. Thereaction module also includes a source of wash solutions in fluidcommunication with the solid phase extraction column for providing washsolutions to elute the remaining impurities on the sorbent, and includesa source of an eluent (e.g., as H₂O/MeCN, or other suitable eluent) influid communication with the solid phase extraction column for elutingthe radiolabeled imaging agent product off the sorbent. The reactionmodule may also include a source of a diluent for the elutedradiolabeled imaging agent.

A formulation module of an apparatus of the invention may be in fluidcommunication with a reaction module and may include a solid phaseextraction cartridge that includes a solid sorbent (e.g., C-18, or othersuitable sorbent) to adsorb the diluted radiolabeled imaging agent, asource of wash solutions (e.g., comprising ascorbic acid, a saltthereof, or other suitable wash solution) in fluid communication withthe solid phase extraction cartridge for providing wash solutions towash off any remaining impurities on the sorbent, and a source ofeluting fluid (e.g., ethanol, or other suitable eluting fluid) in fluidcommunication with the solid phase extraction cartridge for eluting theradiolabeled imaging agent product off the sorbent. The formulationmodule may also include a source of a diluent (e.g., comprising ascorbicacid, a salt thereof, or other suitable diluent), for diluting theeluted radiolabeled imaging agent. The formulation module may also be influid communication with a sterilizing filter (e.g., a Millipore MillexGV PVDF sterilizing filter, or other suitable sterilizing filter).

In a particular embodiment, a cassette is provided for use with anautomated synthesis module, for example, a GE TRACERlab MX synthesismodule. In one embodiment, a cassette comprises a disposable sterilizedassembly of molded stopcock manifolds specifically designed for use withthe automated synthesis module (e.g., GE TRACERlab MX synthesis module).Individual manifolds are connected in a linear or non-linear fashion toform a directional array that dictates the flow path of reagents used inthe preparation of an imaging agent (e.g., imaging agent 1) injection.In some embodiments, the main body of the cassette contains at least onemanifold comprising a plurality of manifold positions (e.g.,stockcocks). For example, the main body may comprise at least one, two,three, four or more, manifolds. The cassette may comprise between 1 to20 manifold positions, between 1 to 15 manifold positions, between 5 and20 manifold positions, between 5 and 15 manifold positions. Each of themanifolds may or may not be symmetrical. In one embodiment, the mainbody of the cassette contains three plastic manifolds each fitted withfive standard molded stopcocks, thereby having a total of 15 totalmanifold positions. Individual stopcocks are adapted with luer fittingsto accommodate solvents, reagents, syringes, tubing required for gas andliquid handling, etc. The stopcocks are adapted for solvents andreagents and may be fitted with plastic spikes upon which inverted punchvials are located, while those featuring tubing and syringes are fittedwith male luer connections according to function. In some embodiments,the cassette comprises a linear arrangement of a plurality of stopcockmanifolds connected one or more of the components selected from thegroup consisting of a gas inlet, anion exchange cartridge, C-18cartridge, syringe, solvent reservoir, reaction vessel, HPLC system,collection vessel, reservoir for solution of ascorbic acid or saltthereof, and exhaust outlet. In some cases, the cassette furthercomprises tubing. In some cases, the cassette further comprising animaging agent synthesis module, wherein the apparatus is fluidicallyconnected to the cassette. In some cases, the apparatus is capablecarrying out the method of synthesizing an imaging agent as describedherein (e.g., a method of synthesizing imaging agent 1).

The cassette configuration required for the preparation of imaging 1injection is depicted in FIG. 8. The following provides a description ofthe attachments to each of the 15 manifold positions: 1) luer connection(2)—gas inlet and [¹⁸O]H₂O recovery; 2) anion exchange cartridge—QMALight; 3) spike connection—MeCN; 4) syringe—empty; 5) spikeconnection—imaging agent precursor 1; 6) luer connection—reactionvessel; 7) HPLC inlet; 8) spike connection—ascorbic acid; 9) luerconnection—collection vessel; 10) syringe—EtOH; 11) luerconnection—final product vial; 12) spike connection—SWFI; 13) spikeconnection—ascorbic acid; 14) syringe—empty; 15) luer connection(2)—reaction vessel and exhaust. Manifold one (stopcocks 1-5) is joinedto manifold two (stopcocks 6-10) using two male luer connections fittedwith a short length of silicon tubing. Manifold two is connected tomanifold three (stopcocks 11-15) using a C-18 Sep-Pak® and theappropriate luer adapters. Individual manifold connections, luerfittings and all silicon tubing are readily available from commercialsuppliers.

In some embodiments, the present invention provides a cassette for thepreparation of an imaging agent comprising the formula:

comprising: (i) a vessel containing an imaging agent precursorcomprising the formula:

and (ii) a conduit for adding a source of ¹⁸F.Pharmaceutical Compositions

Once an imaging agent or an imaging agent precursor has been prepared orobtained, it may be combined with one or more pharmaceuticallyacceptable excipients to form a pharmaceutical composition that issuitable for administering to a subject, including a human. As would beappreciated by one of skill in this art, the excipients may be chosen,for example, based on the route of administration as described below,the agent being delivered, time course of delivery of the agent, and/orthe health/condition of the subject.

Pharmaceutical compositions of the present invention and for use inaccordance with the present invention may include a pharmaceuticallyacceptable excipient or carrier. As used herein, the term“pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose, and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose, andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; detergents such as Tween 80; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

The pharmaceutical compositions of this invention can be administered tohumans and/or to animals, parenterally, intranasally, intraperitoneally,or via a nasal spray. The mode of administration will vary depending onthe intended use, as is well known in the art. Alternatively,formulations of the present invention may be administered parenterallyas injections (intravenous, intramuscular, or subcutaneous). Theseformulations may be prepared by conventional means, and, if desired, thesubject compositions may be mixed with any conventional additive.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Exemplary Kits

In some embodiments, systems, methods, kits, and cassettes kits for thepreparation of an imaging agent (e.g., imaging agent 1) are provided fordetecting, imaging, and/or monitoring myocardial perfusion. In someembodiments, kits for the administration of an imaging agent (e.g.,imaging agent 1) are provided. Kits of the invention may include, forexample, a container comprising an imaging agent, or an imaging agentprecursor, and instructions for use. Kits may include a sterile,non-pyrogenic, formulation comprising a predetermined amount of animaging agent (e.g., imaging agent 1), and optionally other components.In some aspects of the invention, a kit may include one or more syringesthat contain an imaging agent (e.g., imaging agent 1) to be prepared foradministration to a subject. A container that may be used in conjunctionwith an imaging agent (e.g., imaging agent 1) (e.g. to deliver and/oradminister an imaging agent (e.g., imaging agent 1) to a subject) may bea syringe, bottle, vial, tubes, etc. Exemplary syringes that may beincluded in a kit of the invention are syringes lacking an adsorbentplunger tip, such as a 3 or 5 mL NORM-JECT (Henke Sass Wolf, Dudley,Mass.), or other equivalent syringe lacking an adsorbent plunger tip. Animaging agent (e.g., imaging agent 1) may be provided in a kit andadditional preparations before use may optionally include diluting theimaging agent to a usable concentration. Instructions in a kit of theinvention may relate to methods for, methods of diluting the imagingagent, methods of administering the imaging agent to a subject fordiagnostic imaging, or other instructions for use.

In some cases, a kit can also include one or more vials containing adiluent for preparing an imaging agent (e.g., imaging agent 1)composition for administration to a subject (e.g., human). A diluentvial may contain a diluent such as physiological saline, water, bufferedsolution, etc. for diluting an imaging agent (e.g., imaging agent 1).For example, the imaging agent (e.g., imaging agent 1) may be packagedin a kit in a ready-to-inject formulation, or may require somereconstitution or dilution whereby a final composition/formulation forinjection or infusion is prepared.

Instructions in a kit of the invention may also include instructions foradministering the imaging agent (e.g., imaging agent 1) to a subject andmay include information on dosing, timing, stress induction, etc. Forexample, a kit may include an imaging agent described herein, along withinstructions describing the intended application and the properadministration of the agent. As used herein, “instructions” can define acomponent of instruction and/or promotion, and typically involve writteninstructions on or associated with packaging of the invention.Instructions also can include any oral or electronic instructionsprovided in any manner such that a user will clearly recognize that theinstructions are to be associated with the kit, for example, audiovisual(e.g., videotape, DVD, etc.), Internet, and/or web-based communications,etc. The written instructions may be in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which instructions can alsoreflects approval by the agency of manufacture, use or sale for humanadministration. In some cases, the instructions can include instructionsfor mixing a particular amount of the diluent with a particular amountof a concentrated solution of the imaging agent or a solid preparationof the imaging agent, whereby a final formulation for injection orinfusion is prepared for example, such that the resulting solution is ata suitable concentration for administration to a subject (e.g., at aconcentration as described herein). A kit may include a whole treatmentregimen of the inventive compound (e.g., a rest dose and a stress dose).

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing an agent described herein. The agent may bein the form of a liquid, gel or solid (powder). The agent may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include an active agent premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to apatient, such as a syringe, topical application devices, or iv needletubing and bag.

It also will be understood that containers containing the components ofa kit of the invention, whether the container is a bottle, a vial (e.g.,with a septum), an ampoule, an infusion bag, or the like, can includeadditional indicia such as conventional markings that change color whenthe preparation has been autoclaved or otherwise sterilized. A kit ofthe invention may further include other components, such as syringes,labels, vials, tubing, catheters, needles, ports, and the like. In someaspect of the invention, a kit may include a single syringe containingthe imaging agent (e.g., imaging agent 1) sufficient for administrationand in some aspects of the invention a kit may include two separatesyringes, one comprising imaging agent 1 to be administered to a subjectfor rest imaging, and a second syringe comprising imaging agent 1 foradministration to a subject for stress imaging.

Buffers useful in the preparation of imaging agents and kits include,for example, phosphate, citrate, sulfosalicylate, and acetate buffers. Amore complete list can be found in the United States Pharmacopoeia.Lyophilization aids useful in the preparation of imaging agents and kitsinclude, for example, mannitol, lactose, sorbitol, dextran, FICOLL®polymer, and polyvinylpyrrolidine (PVP). Stabilization aids useful inthe preparation of imaging agents and kits include, for example,ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodiummetabisulfite, gentisic acid, and inositol. Solubilization aids usefulin the preparation of imaging agents and kits include, for example,ethanol, glycerin, polyethylene glycol, propylene glycol,polyoxyethylene sorbitan monooleate, sorbitan monoloeate, polysorbates,poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymers(“Pluronics”) and lecithin. In certain embodiments, the solubilizingaids are polyethylene glycol, cyclodextrins, and Pluronics.Bacteriostats useful in the preparation of imaging agents and kitsinclude, for example, benzyl alcohol, benzalkonium chloride,chlorbutanol, and methyl, propyl, or butyl paraben.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are listed here.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in “Organic Chemistry,” Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

As used herein, the term “alkyl” is given its ordinary meaning in theart and refers to the radical of saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In some cases, the alkyl group may be a loweralkyl group, i.e., an alkyl group having 1 to 10 carbon atoms (e.g.,methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, ordecyl, etc.). In some embodiments, a straight chain or branched chainalkyl may have 30 or fewer carbon atoms in its backbone, and, in somecases, 20 or fewer. In some embodiments, a straight chain or branchedchain alkyl may have 12 or fewer carbon atoms in its backbone (e.g.,C₁-C₁₂ for straight chain, C₃-C₁₂ for branched chain), 6 or fewer, or 4or fewer. Likewise, cycloalkyls may have from 3-10 carbon atoms in theirring structure, or 5, 6 or 7 carbons in the ring structure. Examples ofalkyl groups include, but are not limited to, methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, cyclobutyl, hexyl,cyclochexyl, and the like.

The terms “alkenyl” and “alkynyl” are given their ordinary meaning inthe art and refer to unsaturated aliphatic groups analogous in lengthand possible substitution to the alkyls described above, but thatcontain at least one double or triple bond respectively.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-8 aliphatic carbon atoms. In still other embodiments, the alkyl,alkenyl, and alkynyl groups employed in the invention contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-4 carbon atoms.Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl,isobutyl, t-butyl, n-pentyl, sec-pentyl, isopentyl, t-pentyl, n-hexyl,sec-hexyl, moieties and the like, which again, may bear one or moresubstituents. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike. Representative alkynyl groups include, but are not limited to,ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.

The term “cycloalkyl,” as used herein, refers specifically to groupshaving three to ten, preferably three to seven carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic, heteroaliphatic, or hetercyclic moieties, mayoptionally be substituted with substituents including, but not limitedto aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term “heteroalkyl” is given its ordinary meaning in the art andrefers to an alkyl group as described herein in which one or more carbonatoms is replaced by a heteroatom. Suitable heteroatoms include oxygen,sulfur, nitrogen, phosphorus, and the like. Examples of heteroalkylgroups include, but are not limited to, alkoxy, amino, thioester,poly(ethylene glycol), alkyl-substituted amino, tetrahydrofuranyl,piperidinyl, morpholinyl, etc.

The terms “heteroalkenyl” and “heteroalkynyl” are given their ordinarymeaning in the art and refer to unsaturated aliphatic groups analogousin length and possible substitution to the heteroalkyls described above,but that contain at least one double or triple bond respectively.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CHF₂; —CH₂F; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alycyclic, heteroaliphatic, heterocyclic, aryl,heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic,heteroaliphatic, alkylaryl, or alkylheteroaryl substituents describedabove and herein may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and wherein any of the aryl or heteroarylsubstituents described above and herein may be substituted orunsubstituted. Additional examples of generally applicable substituentsare illustrated by the specific embodiments shown in the Examples thatare described herein.

The term “aryl” is given its ordinary meaning in the art and refers toaromatic carbocyclic groups, optionally substituted, having a singlering (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fusedrings in which at least one is aromatic (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl). That is,at least one ring may have a conjugated pi electron system, while other,adjoining rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls. The aryl group may be optionally substituted, asdescribed herein. Substituents include, but are not limited to, any ofthe previously mentioned substitutents, i.e., the substituents recitedfor aliphatic moieties, or for other moieties as disclosed herein,resulting in the formation of a stable compound. In some cases, an arylgroup is a stable mono- or polycyclic unsaturated moieties havingpreferably 3-14 carbon atoms, each of which may be substituted orunsubstituted. “Carbocyclic aryl groups” refer to aryl groups whereinthe ring atoms on the aromatic ring are carbon atoms. Carbocyclic arylgroups include monocyclic carbocyclic aryl groups and polycyclic orfused compounds (e.g., two or more adjacent ring atoms are common to twoadjoining rings) such as naphthyl groups.

The terms “heteroaryl” is given its ordinary meaning in the art andrefers to aryl groups comprising at least one heteroatom as a ring atom.A “heteroaryl” is a stable heterocyclic or polyheterocyclic unsaturatedmoieties having preferably 3-14 carbon atoms, each of which may besubstituted or unsubstituted. Substituents include, but are not limitedto, any of the previously mentioned substitutents, i.e., thesubstituents recited for aliphatic moieties, or for other moieties asdisclosed herein, resulting in the formation of a stable compound. Insome cases, a heteroaryl is a cyclic aromatic radical having from fiveto ten ring atoms of which one ring atom is selected from S, O, and N;zero, one, or two ring atoms are additional heteroatoms independentlyselected from S, O, and N; and the remaining ring atoms are carbon, theradical being joined to the rest of the molecule via any of the ringatoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and thelike.

It will also be appreciated that aryl and heteroaryl moieties, asdefined herein may be attached via an alkyl or heteroalkyl moiety andthus also include -(alkyl)aryl, -(heteroalkyl)aryl,-(heteroalkyl)heteroaryl, and -(heteroalkyl)heteroaryl moieties. Thus,as used herein, the phrases “aryl or heteroaryl moieties” and “aryl,heteroaryl, -(alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)heteroaryl,and -(heteroalkyl)heteroaryl” are interchangeable. Substituents include,but are not limited to, any of the previously mentioned substituents,i.e., the substituents recited for aliphatic moieties, or for othermoieties as disclosed herein, resulting in the formation of a stablecompound.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one or more of the hydrogen atomsthereon independently with any one or more of the following moietiesincluding, but not limited to: aliphatic; alicyclic; heteroaliphatic;heterocyclic; aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl;heteroalkylaryl; alkylheteroaryl; heteroalkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH; —NO₂; —CN; —CF₃;—CH₂F; —CHF₂; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃;—C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x);—OCON(R_(x))₂; —N(R_(x))₂; —S(O)R_(x); —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alicyclic, heteroaliphatic, heterocyclic,aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl or heteroalkylheteroaryl, wherein any of the aliphatic,alicyclic, heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aromatic, heteroaromatic, aryl, heteroaryl,-(alkyl)aryl or -(alkyl)heteroaryl substituents described above andherein may be substituted or unsubstituted. Additionally, it will beappreciated, that any two adjacent groups taken together may represent a4, 5, 6, or 7-membered substituted or unsubstituted alicyclic orheterocyclic moiety. Additional examples of generally applicablesubstituents are illustrated by the specific embodiments describedherein.

The term “heterocycle” is given its ordinary meaning in the art andrefers to refer to cyclic groups containing at least one heteroatom as aring atom, in some cases, 1 to 3 heteroatoms as ring atoms, with theremainder of the ring atoms being carbon atoms. Suitable heteroatomsinclude oxygen, sulfur, nitrogen, phosphorus, and the like. In somecases, the heterocycle may be 3- to 10-membered ring structures or 3- to7-membered rings, whose ring structures include one to four heteroatoms.

The term “heterocycle” may include heteroaryl groups, saturatedheterocycles (e.g., cycloheteroalkyl) groups, or combinations thereof.The heterocycle may be a saturated molecule, or may comprise one or moredouble bonds. In some cases, the heterocycle is a nitrogen heterocycle,wherein at least one ring comprises at least one nitrogen ring atom. Theheterocycles may be fused to other rings to form a polycylicheterocycle. The heterocycle may also be fused to a spirocyclic group.In some cases, the heterocycle may be attached to a compound via anitrogen or a carbon atom in the ring.

Heterocycles include, for example, thiophene, benzothiophene,thianthrene, furan, tetrahydrofuran, pyran, isobenzofuran, chromene,xanthene, phenoxathiin, pyrrole, dihydropyrrole, pyrrolidine, imidazole,pyrazole, pyrazine, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,triazole, tetrazole, oxazole, isoxazole, thiazole, isothiazole,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, oxazine, piperidine, homopiperidine(hexamnethyleneimine), piperazine (e.g., N-methyl piperazine),morpholine, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, other saturated and/or unsaturated derivativesthereof, and the like. The heterocyclic ring can be optionallysubstituted at one or more positions with such substituents as describedherein. In some cases, the heterocycle may be bonded to a compound via aheteroatom ring atom (e.g., nitrogen). In some cases, the heterocyclemay be bonded to a compound via a carbon ring atom. In some cases, theheterocycle is pyridine, imidazole, pyrazine, pyrimidine, pyridazine,acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline,benzoquinoline, benzoisoquinoline, phenanthridine-1,9-diamine, or thelike.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “amino,” as used herein, refers to a primary (—NH₂), secondary(—NHR_(x)), tertiary (—NR_(x)R_(y)), or quaternary(—N⁺R_(x)R_(y)R_(z))amine, where R_(x), R_(y) and R_(z) areindependently an aliphatic, alicyclic, heteroaliphatic, heterocyclic,aryl, or heteroaryl moiety, as defined herein. Examples of amino groupsinclude, but are not limited to, methylamino, dimethylamino, ethylamino,diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino,piperidino, trimethylamino, and propylamino.

The term “alkyne” is given its ordinary meaning in the art and refers tobranched or unbranched unsaturated hydrocarbon groups containing atleast one triple bond. Non-limiting examples of alkynes includeacetylene, propyne, 1-butyne, 2-butyne, and the like. The alkyne groupmay be substituted and/or have one or more hydrogen atoms replaced witha functional group, such as a hydroxyl, halogen, alkoxy, and/or arylgroup.

The term “alkoxy” (or “alkyloxy”), or “thioalkyl” as used herein refersto an alkyl group, as previously defined, attached to the parentmolecular moiety through an oxygen atom or through a sulfur atom. Incertain embodiments, the alkyl group contains 1-20 aliphatic carbonatoms. In certain other embodiments, the alkyl group contains 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-8 aliphaticcarbon atoms. In still other embodiments, the alkyl group contains 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl groupcontains 1-4 aliphatic carbon atoms. Examples of alkoxy, include but arenot limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,t-butoxy, neopentoxy and n-hexoxy. Examples of thioalkyl include, butare not limited to, methylthio, ethylthio, propylthio, isopropylthio,n-butylthio, and the like.

The term “aryloxy” refers to the group, —O-aryl. The term “acyloxy”refers to the group, —O-acyl.

The term “alkoxyalkyl” refers to an alkyl group substituted with atleast one alkoxy group (e.g., one, two, three, or more, alkoxy groups).For example, an alkoxyalkyl group may be —(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl),optionally substituted. In some cases, the alkoxyalkyl group may beoptionally substituted with another alkyoxyalkyl group (e.g.,—(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl), optionally substituted.

It will be appreciated that the above groups and/or compounds, asdescribed herein, may be optionally substituted with any number ofsubstituents or functional moieties. That is, any of the above groupsmay be optionally substituted. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds, “permissible” being in the context of the chemical rules ofvalence known to those of ordinary skill in the art. In general, theterm “substituted” whether preceded by the term “optionally” or not, andsubstituents contained in formulas of this invention, refer to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. When more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. It will be understood that “substituted”also includes that the substitution results in a stable compound, e.g.,which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc. In some cases,“substituted” may generally refer to replacement of a hydrogen with asubstituent as described herein. However, “substituted,” as used herein,does not encompass replacement and/or alteration of a key functionalgroup by which a molecule is identified, e.g., such that the“substituted” functional group becomes, through substitution, adifferent functional group. For example, a “substituted phenyl group”must still comprise the phenyl moiety and can not be modified bysubstitution, in this definition, to become, e.g., a pyridine ring. In abroad aspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnonaromatic substituents of organic compounds. Illustrative substituentsinclude, for example, those described herein. The permissiblesubstituents can be one or more and the same or different forappropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valencies of the heteroatoms. Furthermore, this invention isnot intended to be limited in any manner by the permissible substituentsof organic compounds. Combinations of substituents and variablesenvisioned by this invention are preferably those that result in theformation of stable compounds useful for the formation of an imagingagent or an imaging agent precursor. The term “stable,” as used herein,preferably refers to compounds which possess stability sufficient toallow manufacture and which maintain the integrity of the compound for asufficient period of time to be detected and preferably for a sufficientperiod of time to be useful for the purposes detailed herein.

Examples of substituents include, but are not limited to, halogen,azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromaticmoieties, —CF₃, —CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl,heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, amino, halide,alkylthio, oxo, acylalkyl, carboxy esters, -carboxamido, acyloxy,aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl,arylamino, aralkylamino, alkylsulfonyl, -carboxamidoalkylaryl,-carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy-,aminocarboxamidoalkyl-, cyano, alkoxyalkyl, perhaloalkyl,arylalkyloxyalkyl, and the like.

As used herein, the term “determining” generally refers to the analysisof a species or signal, for example, quantitatively or qualitatively,and/or the detection of the presence or absence of the species orsignals. “Determining” may also refer to the analysis of an interactionbetween two or more species or signals, for example, quantitatively orqualitatively, and/or by detecting the presence or absence of theinteraction.

As used herein the term “acquiring” an image means obtaining an image.

The term “diagnostic imaging,” as used herein, refers to a procedureused to detect an imaging agent.

A “diagnostic kit” or “kit” comprises a collection of components, termedthe formulation, in one or more vials, which are used in a clinical orpharmacy setting to synthesize diagnostic radiopharmaceuticals. Forexample, the kit may be used by the practicing end user in a clinical orpharmacy setting to synthesize and/or use diagnosticradiopharmaceuticals. In some embodiments, the kit may provide all therequisite components to synthesize and/or use the diagnosticpharmaceutical except those that are commonly available to thepracticing end user, such as water or saline for injection, a solutionof the radionuclide, equipment for processing the kit during thesynthesis and manipulation of the radiopharmaceutical, if required,equipment necessary for administering the radiopharmaceutical to thesubject such as syringes, shielding, imaging equipment, and the like. Insome embodiments, imaging agents may be provided to the end user intheir final form in a formulation contained typically in one vial orsyringe, as either a lyophilized solid or an aqueous solution.

As used herein, a “portion of a subject” refers to a particular regionof a subject, location of the subject. For example, a portion of asubject may be the brain, heart, vasculature, cardiac vessels, of asubject.

As used herein a “session” of testing may be a single testing protocolthat a subject undergoes. In some cases a session may includerest/stress imaging protocol; stress/rest imaging protocol; rest onlyimaging protocol; or a stress only imaging protocol. A session oftesting can take place in less than 24 hours or less than 48 hour.

As used herein, the term “subject” refers to a human or non-human mammalor animal. Non-human mammals include livestock animals, companionanimals, laboratory animals, and non-human primates. Non-human subjectsalso specifically include, without limitation, horses, cows, pigs,goats, dogs, cats, mice, rats, guinea pigs, gerbils, hamsters, mink, andrabbits. In some embodiments of the invention, a subject is referred toas a “patient.” In some embodiments, a patient or subject may be underthe care of a physician or other health care worker, including, but notlimited to, someone who has consulted with, received advice from orreceived a prescription or other recommendation from a physician orother health care worker.

Any of the compounds described herein may be in a variety of forms, suchas, but not limited to, salts, solvates, hydrates, tautomers, andisomers.

In certain embodiments, the imaging agent is a pharmaceuticallyacceptable salt of the imaging agent. The term “pharmaceuticallyacceptable salt” as used herein refers to those salts which are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al., describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 1977, 66, 1-19, incorporated herein by reference.Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

In certain embodiments, the compound is in the form of a hydrate orsolvate. The term “hydrate” as used herein refers to a compoundnon-covalently associated with one or more molecules of water. Likewise,the term “solvate” refers to a compound non-covalently associated withone or more molecules of an organic solvent.

In certain embodiments, the compound described herein may exist invarious tautomeric forms. The term “tautomer” as used herein includestwo or more interconvertable compounds resulting from at least oneformal migration of a hydrogen atom and at least one change in valency(e.g., a single bond to a double bond, a triple bond to a single bond,or vice versa). The exact ratio of the tautomers depends on severalfactors, including temperature, solvent, and pH. Tautomerizations (i.e.,the reaction providing a tautomeric pair) may catalyzed by acid or base.Exemplary tautomerizations include keto-to-enol; amide-to-imide;lactam-to-lactim; enamine-to-imine; and enamine-to-(a different) enaminetautomerizations.

In certain embodiments, the compounds described herein may exist invarious isomeric forms. The term “isomer” as used herein includes anyand all geometric isomers and stereoisomers (e.g., enantiomers,diasteromers, etc.). For example, “isomer” include cis- andtrans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers,(D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixturesthereof, as falling within the scope of the invention. For instance, anisomer/enantiomer may, in some embodiments, be provided substantiallyfree of the corresponding enantiomer, and may also be referred to as“optically enriched.” “Optically-enriched,” as used herein, means thatthe compound is made up of a significantly greater proportion of oneenantiomer. In certain embodiments the compound of the present inventionis made up of at least about 90% by weight of a preferred enantiomer. Inother embodiments the compound is made up of at least about 95%, 98%, or99% by weight of a preferred enantiomer. Preferred enantiomers may beisolated from racemic mixtures by any method known to those skilled inthe art, including chiral high pressure liquid chromatography (HPLC) andthe formation and crystallization of chiral salts or prepared byasymmetric syntheses. See, for example, Jacques, et al., Enantiomers,Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen,S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistryof Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972).

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 Synthesis of 4-(2-hydroxyethoxymethyl)benzoic acidmethyl ester

To a two-neck round bottom flask, which was equipped with a Dewarcondenser, a solution of 4-hydroxymethylbenzoic acid methyl ester (2.50g, 0.015 mol) in anhydrous dichloromethane (30 mL) was cooled to −10° C.in a salt/ice bath. Ethylene oxide (1.10 mL) was added to the cooledstiffing solution dropwise followed by the addition of boron trifluorideetherate (0.51 ml). The reaction mixture was stirred for 45 minutes andthen warmed to room temperature for 30 minutes to boil off any excess ofethylene oxide from the reaction mixture. The reaction mixture was thendiluted with brine. The aqueous layer was extracted with dichloromethane(3 times). All of the organic layers were combined, dried over Na₂SO₄,filtered, and concentrated to provide an oil. The crude material waspurified using silica gel chromatography (4:1 pentane:ethyl acetate) toprovide the desired product (537 mg, 2.56 mmol) in 17% yield. ¹H(CDCl₃8.36, 600 MHz): δ (2H, d, J=8.4 Hz), 7.41 (2H, d, J=8.5 Hz), 4.62(3H, s), 3.92 (2H, s), 3.78 (m, 2H), 3.63 (2H, m); ¹³C (CDCl₃167.1,143.5, 130.0, 129.8, 127.5, 72.9, 72.0, 150 MHz): δ 62.1, 52.3.

Example 2 Synthesis of4-[2-(t-butyldimethylsilanyloxy)ethoxymethyl]benzoic acid methyl ester

To a solution of the product of Example 1 (544.5 mg, 2.59 mmol) inanhydrous DMF (26 mL) was added imidazole (264 mg, 3.89 mmol) andTBDMS-Cl (586 mg, 3.89 mmol). The reaction mixture stirred at roomtemperature overnight and was quenched with water. The aqueous layer wasextracted with ethyl acetate (3×). All combined organic layers weredried over Na₂SO₄, filtered, and concentrated. The crude material waspurified using silica gel chromatography (4:1 pentane:ethyl acetate) toprovide the desired product (677.5 mg, 2.19 mmol) in 84% yield. ¹H(CDCl₃ 8.01, 600 MHz): δ (2H, d, J=8.3 Hz), 7.42 (2H, d, J=8.4 Hz), 4.63(2H, s), 3.91 (2H, s), 3.82 (2H, t, J=5.0), 3.58 (2H, t, J=5.1 Hz), 0.91(9H, s), 0.07 (6H, s); ¹³C (CDCl₃166.5, 143.5, 129.2, 128.8, 126.5,72.1, 71.6, 150 MHz): δ 62.3, 51.5, 25.4, 17.9, −5.8.

Example 3 Synthesis of{4-[2-(t-butyldimethylsilanyloxy)ethoxymethyl]phenyl}methanol

To a solution of the product of Example 2 (670 mg, 2.18 mmol) dissolvedin anhydrous THF (22 mL) was added a solution of LAH (1.0 M solution inTHF, 2.18 mL, 2.18 mmol) dropwise. After completion of addition thereaction mixture was stirred at room temperature for 3 hours. Thereaction mixture was diluted with water. The aqueous layer was extractedwith ethyl acetate (3×). All combined organic layers were dried overNa₂SO₄, filtered, and concentrated to provide an oil (587 mg, 1.98mmol), which was used in the next step without any further purification(91% yield). ¹H (CDCl₃ 7.34 (4H, s), 4.68 (2H, s), 4.57 (2H, s), 3.80,600 MHz): δ (2H, t, J=5.2 Hz), 3.56 (2H, t, J=5.3 Hz), 1.69 (1H, br s),0.90 (9H, s), 0.07 (6H, s); ¹³C (CDCl₃ 140.4, 138.3, 128.0, 127.2, 73.2,71.9, 65.4, 150 MHz): δ 63.0, 26.2, 18.6, −5.0.

Example 4 Synthesis of2-t-butyl-5-{4-[2-(t-butyldimethylsilanyloxy)ethoxymethyl]benzyloxy}-4-chloro-2H-pyridazin-3-one

To solution of the product of Example 3 (437 mg, 1.48 mmol) and2-t-butyl-4-chloro-5-hydroxy-2H-pyridazin-3-one (250 mg, 1.23 mmol)dissolved in anhydrous THF (12 mL) was added solid PPh₃ (485 mg, 1.85mmol) and diisopropyl azodicarboxylate (DIAD, 0.358 mL, 1.85 mmol).After completion of addition the reaction mixture continued to stir atroom temperature. After 20 hours, the reaction mixture was diluted withwater. The aqueous layer was separated and extracted with ethyl acetate(3×). All combined organic layers were dried over Na₂SO₄, filtered, andconcentrated to provide an oil. The crude material was purified usingsilica gel chromatography (4:1 pentane:ethyl acetate) to provide thedesired product 528 mg, 1.10 mmol) in 89% yield. ¹H (CDCl₃ 7.70 (1H, s),7.38 (4H, m), 5.30 (2H, s), 4.58, 600 MHz): δ (2H, s), 3.80 (2H, t,J=5.4 Hz), 3.57 (2H, t, J=5.4 Hz), 1.63 (9H, br s), 0.90 (9H, s), 0.07(6H, s); ¹³C (CDCl₃159.0, 153.7, 138.8, 134.4, 128.3, 127.3, 150 MHz): δ125.1, 118.5, 72.8, 71.7, 71.6, 66.4, 61.9, 29.7, 27.9, 25.6, −5.1.;HRMS calcd for C₂₄H₃₇ClN₂O₄Si: 481.228389. found 481.2282.

Example 5 Synthesis of2-t-butyl-4-chloro-5-[4-(2-hydroxyethoxymethyl)benzyloxy]-2H-pyridazin-3-one

To a solution of the product of Example 4 (528 mg, 1.09 mmol) dissolvedin anhydrous THF (11 mL) was added a solution of TBAF (1.0 M solution inTHF, 1.65 mL, 1.65 mmol) dropwise. After completion of addition thereaction was stirred at room temperature for 1 hour and then quenchedwith water. The aqueous layer was separated and extracted with ethylacetate (3×). All combined organic layers were dried over Na₂SO₄,filtered, and concentrated to provide an oil. The crude material waspurified using silica gel chromatography (4:1 hexanes:ethyl acetate) toprovide the desired product (311 mg, 0.850 mmol) in 78% yield. ¹H(CDCl₃, 600 MHz): δ 7.70 (1H, s), 7.38 (4H, m), 5.30 (2H, s), 4.56 (2H,s), 3.76 (2H, t, J=4.9 Hz), 3.60 (2H, t, J=4.8 Hz), 2.00 (1H, br s),1.61 (9H, br s); ¹³C (CDCl₃159.0, 153.6, 150 MHz): δ 138.8, 134.4,128.2, 127.2, 125.1, 118.3, 72.8, 71.6, 71.6, 66.4, 61.9, 27.8; HRMScalcd for C₁₈H₂₃ClN₂O₄: 367.141911. found 367.1419.

Example 6 Synthesis of toluene-4-sulfonic acid2-[4-(1-t-butyl-5-chloro-6-oxo-1,6-dihydro-pyridazin-4-yloxymethyl)-benzyloxy]-ethylester

To a solution of the product of Example 5 (200 mg, 0.546 mmol) dissolvedin anhydrous dichloromethane (5.50 mL) was added TsCl (125 mg, 0.656mmol), DMAP (100 mg, 0.819 mmol) and triethylamine (0.091 mL, 0.656mmol). The reaction mixture continued stirring at room temperature.After 22 hours the reaction mixture was diluted with water. The aqueouslayer was separated and extracted with ethyl acetate (3×). All combinedorganic layers were dried over Na₂SO₄, filtered, and concentrated toprovide an oil. The crude material was purified using silica gelchromatography (3:2 pentane:ethyl acetate) to provide the desiredproduct (232 mg, 0.447 mmol) in 82% yield. ¹H (CDCl₃7.79, 600 MHz): δ(2H, d, J=8.3 Hz), 7.71 (1H, s), 7.38 (2H, d, J=8.2 Hz), 7.32 (4H, m),5.30 (2H, s), 4.50 (2H, s), 4.21 (2H, m), 3.69 (2H, m), 2.43 (3H, s),1.63 (9H, br s); ¹³C (CDCl₃ 159.0, 153.7, 144.8, 138.8, 150 MHz): δ134.4, 133.1, 129.8, 128.1, 128.0, 127.2, 125.1, 118.4, 72.8, 71.7,69.2, 67.8, 66.4, 27.9, 21.6; HRMS calcd for C₂₅H₂₉ClN₂O₆: 521.150762.found 521.1503.

Example 7 Preparation of [¹⁸F]fluoride

[¹⁸F]Fluoride was produced by proton bombardment of [¹⁸O]H₂O in acyclotron; the nuclear chemical transformation is shown below and may besummarized as ¹⁸O (p,n)¹⁸F. For purposes of the bombardment, thechemical form of the ¹⁸O is H₂ ¹⁸O. The chemical form of the resulting¹⁸F is fluoride ion.¹⁸O+proton→¹⁸F+neutron

According to established industry procedures, [¹⁸O]H₂O (2-3 mL) housedwithin a tantalum target body using Havar® foil, was bombarded with 11MeV protons (nominal energy); where the proton threshold energy for thereaction is 2.57 MeV and the energy of maximum cross section is 5 MeV.Target volume, bombardment time and proton energy each may be adjustedto manage the quantity of [¹⁸F]fluoride produced.

Example 8 Preparation of Imaging Agent Precursor 1 AcetontrileConcentrate

Imaging agent precursor 1 (20.4 g, 39.2 mmol), as shown in FIG. 1, wasdissolved in anhydrous MeCN (3400 mL) then transferred through anOpticap XL2 Durapore filter (0.2 μm) into 5 mL glass vials; 2.0 mL fillvolume. The vials were then fitted with rubber septa, sealed with analuminum crimp and stored at ambient temperature prior to use.

Example 9 General Preparation of Imaging Agent 1

The following example describes a general procedure for synthesizingimaging agent 1, as shown in FIG. 1. Aqueous [¹⁸F]fluoride, as preparedin Example 7, was transferred from the cyclotron to a synthesis module,then filtered through an anion exchange column to remove unreacted[¹⁸O]H₂O; [¹⁸F]fluoride was retained within the cationic resin matrix.The column was then washed with aqueous Et₄NHCO₃ with transfer to thereaction vessel. The resulting solution was diluted with MeCN thenconcentrated to dryness using elevated temperature and reduced pressure.The mixture of anhydrous [¹⁸F]Et₄NF and and Et₄NHCO₃ thus obtained wastreated with the acetonitrile solution of imaging agent precursor 1, asprepared in Example 8, then warmed to 90-100° C. and maintained 10-20min. After cooling, the solution was diluted with H₂O then directlypurified by HPLC on a Waters Xterra MS C18 column using a H₂O/MeCNeluent. The main product peak was collected, diluted with ascorbic acidthen transferred to the formulation module. In another case, similarsteps and conditions were employed as above except the solution waswarmed to 85-120° C. and maintained 5-20 mM, followed by cooling anddiluting with 1:1 H₂O/MeCN.

Example 10 Preparation of imaging agent 1 using the Explora RN SynthesisModule

The product of Example 7 was transferred from cyclotron to the synthesismodule then filtered through an anion exchange column to removeunreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within the cationic resinmatrix. The column was then washed with Et₄NHCO₃ (5.75 μmol; 0.500 mL ofa 11.5 mM solution in H₂O) with transfer to the reaction vessel. Theresulting solution was diluted with MeCN (0.500 mL) then concentrated todryness; 150 mm Hg at 115° C. for 4 mM The mixture of anhydrous[¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained was treated with the product ofExample 8 (11.5 μmol; 1.00 mL of a 11.5 mM solution in MeCN) then warmedto 90° C. and maintained 20 mM After cooling to 35° C., the solution wasdiluted with H₂O (1.00 mL) then directly purified by HPLC on a WatersXterra MS C18 column (10 μm; 10×250 mm) using a 45:55 H₂O/MeCN eluent ata flow rate of 5 mL/min. The main product peak eluting at 11 mM wascollected, diluted with ascorbic acid (10 mL of a 0.28 M solution inH₂O; pH 2) then transferred to the formulation module; 58% decaycorrected radiochemical yield.

In another case, similar steps and conditions were employed as aboveexcept the Et₄NHCO₃ was 11.5 μmol (0.500 mL of a 23.0 mM solution inH₂O); the solution was concentrated to dryness at 280 mbar, 95-115° C.,4 mM; the mixture of anhydrous [¹⁸F]Et₄NF and Et₄NHCO₃ treated with theproduct of Example 8 was warmed to 90° C. and maintained 10 mM; and theproduct had 61% decay corrected radiochemical yield.

Example 11a Preparation of Imaging Agent 1 Using the Eckhert & ZieglerModular-Lab Synthesis Module

The product of Example 7 was transferred from cyclotron to the synthesismodule then filtered through an anion exchange column to removeunreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within the cationic resinmatrix. The column was then washed with Et₄NHCO₃ (11.5 μmol; 0.500 mL ofa 23.0 mM solution in H₂O) with transfer to the reaction vessel. Theresulting solution was diluted with MeCN (0.500 mL) then concentrated todryness; 375 mm Hg at 115° C. for 10 min. The mixture of anhydrous[¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained was treated with the product ofExample 8 (11.5 μmol; 1.00 mL of a 11.5 mM solution in MeCN) then warmedto 110° C. and maintained 10 min. After cooling to 20° C., the solutionwas diluted with H₂O (1.00 mL) then directly purified by HPLC on aWaters Xterra MS C18 column (10 μm; 10×250 mm) using a 45:55 H₂O/MeCNeluent at a flow rate of 5 mL/min. The main product peak eluting at 11min was collected, diluted with ascorbic acid (10 mL of a 0.28 Msolution in H₂O; pH 2) then transferred to the formulation module; 68%decay corrected radiochemical yield.

In another case, similar steps and conditions were employed as aboveexcept the resulting solution was concentrated to dryness a 400 mbar,110-150° C., 10 min; the mixture of anhydrous [¹⁸F]Et₄NF and Et₄NHCO₃treated with the product of Example 8 was warmed to 120° C. andmaintained 10 min; and the cooling was conducted at 35° C.

Example 11b Preparation of Imaging Agent 1 using the Explora GNSynthesis Module

The product of Example 7 was transferred from cyclotron to the synthesismodule then filtered through an anion exchange column to removeunreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within the cationic resinmatrix. The column was then washed with Et₄NHCO₃ (11.5 μmol; 1.00 mL ofa 11.5 mM solution in H₂O) with transfer to the reaction vessel. Theresulting solution was diluted with MeCN (1.00 mL) then concentrated todryness; 110-115° C. Additional MeCN (1.50 mL) was then added and thesolution concentrated to dryness once again. The mixture of anhydrous[¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained was treated with the product ofExample 8 (11.5 μmol; 1.00 mL of a 11.5 mM solution in MeCN) then warmedto 120° C. and maintained 10 min After cooling to 60° C., the solutionwas diluted with H₂O/MeCN (3.00 mL; 2:1 v/v) then directly purified byHPLC on a Waters Xterra MS C18 column (10 μm; 10×250 mm) using a 45:55H₂O/MeCN eluent at a flow rate of 5 mL/min. The main product peakeluting at 11 min was collected, diluted with ascorbic acid (10 mL of a0.28 M solution in H₂O; pH 2) then transferred to the formulationmodule; 68% decay corrected radiochemical yield.

Example 11c Preparation of Imaging Agent 1 Using the GE TRACERLab MXSynthesis Module

The product of Example 7 was transferred from cyclotron to the synthesismodule then filtered through an anion exchange column to removeunreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within the cationic resinmatrix. The column was then washed with Et₄NHCO₃ (23.0 μmol; 0.500 mL ofa 46.0 mM solution in 1:1 H₂O/MeCN) with transfer to the reactionvessel. The resulting solution was diluted with MeCN then concentratedto dryness; 150 mbar, 105° C., 8 min Additional MeCN was then added andthe drying process repeated; the process of MeCN addition followed byevaporation was repeated three times. The mixture of anhydrous[¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained was treated with the product ofExample 8 (23.0 μmol; 2.00 mL of a 11.5 mM solution in MeCN) then warmedto 85° C. and maintained 10 min. The resulting solution was then dilutedwith H₂O (2.00 mL) and directly purified by HPLC on a Waters Xterra MSC18 column (10 μm; 10×250 mm) using a 45:55 H₂O/MeCN eluent at a flowrate of 5 mL/min. The main product peak eluting at 11 min was collected,diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂O; pH 2)then transferred to the formulation module; 63% decay correctedradiochemical yield.

Example 12 Solvent Exchange Process

The product of Example 10 or 11 was transferred from purification to theformulation module then filtered through a C18 Sep-Pak® cartridge toremove MeCN; Imaging agent 1 was retained within the C18 resin matrixand the filtrate discarded. The cartridge was successively washed withascorbic acid (10 mL of a 0.28 M solution in H₂O; pH 2), the filtratediscarded, then absolute EtOH (0.50 mL), and the filtrate collected. Theethanol concentrate of imaging agent 1 thus obtained was further dilutedwith ascorbic acid (10.0 mL of a 0.28 M solution in H₂O) in preparationfor final aseptic filtration.

Example 13 Aseptic Filtration Process

The final product vial assembly was constructed from the followingpre-sterilized components: one 30 mL product vial, one Millipore MillexGV4 venting filter (0.22 μm×4 mm), one tuberculin syringe (1 mL) and oneinsulin syringe (0.5 mL). The product of Example 12 was then transferredfrom formulation to the final product vial assembly through a MilliporeMillex GV PVDF sterilizing filter (0.22 μm×13 mm) Quality controlsamples are then removed, using the syringe assemblies, to complete allproduct release requirements.

Example 14

Upon evaluation of several experimental parameters in the nucleophilicfluorination of imaging agent precursor 1 (FIG. 1) usingK₂CO₃/Kryptofix® 222 overall reaction complexity was shown to increasewith added K₂CO₃; comparable fluorination efficiency was observedregardless of reagent stoichiometry. Elevated base (e.g., carbonate)levels were simply correlated to unproductive consumption of startingmaterial (e.g., imaging agent precursor). Substitution of K₂CO₃ withKHCO₃ resulted in considerable improvement of both fluorinationefficiency and starting material integrity. The solution pH remaineduniform regardless of base identity and reagent stoichiometry; thepresence or absence of Kryptofix® 222 determines global solution pH. Thefluorination efficiency remained stable regardless of reagentstoichiometry, indicating a more complex role of added base within thereaction coordinate.

FIG. 2 shows various possible reaction pathways, which tracesunproductive consumption of starting material to a series ofbase-mediated hydrolysis reactions and dimerization events. Variabletime and temperature experiments confirmed the comparable rates ofhydrolysis and fluorination in the nucleophilic fluorination reactionshown in FIG. 1, using the K₂CO₃/Kryptofix® 222 in the presence ofK₂CO₃. Thus, reaction conditions which activate larger differentialrates of fluorination are desired in order to advance a more efficientand chemoselective process; that is, a decreased rate of hydrolysisand/or increased rate of fluorination.

As noted above, K₂CO₃ did little to enhance fluorination over baselinelevels and served primarily a detrimental role in the reaction. Incontrast, added KHCO₃ produced a marked increase in fluorination overthe same dynamic range, while decomposition pathways remained poorlydifferentiated. These facts, coupled with the knowledge that [¹⁸F]NaFexchange with tetraalkylammonium cations is known to directly produce ahighly active nucleophilic fluoride source, led to the investigation ofa series of commercially available salts in an effort to identifyrelated counterion affects that amplify the rate of fluorination (e.g.,see FIG. 1).

A series of different bases was used in the nucleophilic fluorination ofa tosylate precursor using TBAF as a source of fluoride (shown above),according to the following procedure. A 2 mL glass vial was charged withboth Bu₄NF (1.15 μmol; 13.4 μL of a 85.9 mM solution in H₂O) andBu₄NHCO₃ (10.4 μmol; 138 μL of a 75.0 mM solution in H₂O), then warmedto 95° C. and maintained 10 min under a stream of dry nitrogen. Theresulting solid mixture was treated with the product of Example 8 (11.5μmol; 1.00 mL of a 11.5 mM solution in MeCN) then warmed to 90° C. andmaintained 10 min. After cooling to 22° C., the resulting solution wasdiluted with H₂O then directly analyzed by HPLC on a Zorbax SB-C18column (4.6×50 mm) using a H₂O/MeCN gradient containing 0.1% HCO₂H witha flow rate of 1.00 mL/min. The reaction yield was then calculatedthrough comparison of the integrated peak area for the product in thecrude reaction mixture to that of the authentic standard product (Table1); results obtained through substitution of several alternate saltforms are also provided for comparison.

An enhancement of fluorination efficiency was observed in the presenceof bicarbonate anion. Additionally, a modest dependence on size of thealkyl substituent was observed when R=methyl→ethyl→butyl (data notshown).

A ˜1.5-fold improvement in yield was observed using the KF-Kryptofix®222 method when changing from no added salt, to one equivalent potassiumcarbonate, to one equivalent potassium bicarbonate.

TABLE 1 Comparison of salt form identity and fluorination yield. saltform % yield bicarbonate 81.4 hydroxide 35.5 acetate 2.8 lactate 38.7trifluoroacetate 3.7 methanesulfonate 39.6 p-toluenesulfonate 15.0nitrate 45.1 iodide 44.6 bisulfate <2% none 44.1

Additionally, the amount of salt additive was varied, relative to theamount of starting material (e.g., imaging agent precursor), in order toinvestigate the effect of salt additive concentration on the reaction.FIG. 9 shows (A) a graph illustrating the changes in productdistribution as a function of molar concentration of bicarbonate saltand (B) a graph illustrating the product distribution as a function ofreaction time. Investigation of the salt stoichiometry revealed that 25mol % (or 0.25 equivalents, relative to the imaging agent precursor) oftetraalkylammonium bicarbonate was needed for complete conversion andunproductive consumption of starting material occurred with increasingbase concentration revealing an optimum stoichiometry range for themodified reaction conditions. Related studies directed towarddetermination of the optimal precursor concentration revealed a ratherdistinct concentration threshold. FIG. 9C illustrates a threshold of >3mg/ml.

The use of tetraalkylammonium bicarbonate as an additive in the absenceof Kryptofix® 222 during nucleophilic fluorination resulted in rapidconversion to the desired product and significantly improvedchemoselectivity toward fluorination, relative to the use ofK₂CO₃/Kryptofix® 222 method. A detailed evaluation of crude reactionmixtures revealed a dramatic reduction in overall decomposition rateswhen tetraalkylammonium bicarbonate was used, as evidenced by theabsence of four hydrolytic impurities present when K₂CO₃/Kryptofix® 222was used. Without wishing to be bound by theory, this may be attributedto the fact that the use of a tetraalkylammonium bicarbonate allows thereaction to be conducted at a lower absolute pH (e.g., a pH of about5-6).

Example 15

The following example investigates the effect of the presence ofpotassium carbonate in a nucleophilic fluorination reaction. A yield of36% is obtained in the presence of potassium carbonate, while a yield of35% is obtained in the absence of potassium carbonate.

Example 16

The following example describes the effect that different salt additivesmay have on nucleophilic fluorination. A yield of 35% is obtained in thepresence of potassium carbonate, while a yield of 71% is obtained in thepresence of potassium bicarbonate.

Example 17

The following example describes the results obtained using differentfluoride sources in a nucleophilic fluorination reaction. A yield of 71%is obtained in the presence of KF/Kryptofix® 222, while a yield of 83%is obtained in the presence of tetrabutylammonium fluoride.

Example 18

The following example describes the results obtained using differentbases in a nucleophilic fluorination reaction utilizingtetrabutylammonium fluoride as the fluoride salt. A yield of 83% isobtained in the presence of the bicarbonate base, while a yield of 36%is obtained in the presence of the hydroxide base.

Example 19

The following describes a comparison of imaging agent 1 and ⁸²Rb PETversus SPECT for detection of myocardial ischemia. In preclinicalstudies, myocardial uptake of imaging agent 1 exhibits a strongerrelationship with myocardial blood flow across the range of achievableflow than ²⁰¹Tl, ^(99m)Tc sestamibi and ⁸²Rb. The following experimentswere conducted to determine if the improved extraction and retention ofimaging agent 1 would result in a greater difference between PET andSPECT ischemia detection by imaging agent 1 versus ⁸²Rb.

Methods: Twenty-six patients (20 men) who underwent ^(99m)Tc sestamibiSPECT and imaging agent 1 PET within 6 months at a single center in aphase II clinical trial were compared to 23 patients (matched by summeddifference score (SDS) on SPECT) who underwent ^(99m)Tc sestamibi SPECTand ⁸²Rb PET (25-50 mCi) within 6 months without change in clinicalstate. PET was performed with imaging agent 1 at rest (2.3-3.9 mCi)followed 60 min or 24 h later with exercise or adenosine stress (7.3-8.6mCi). Perfusion defects on SPECT and PET were assessed bycomputer-assisted visual interpretation, using the standard 17 segment,5 point-scoring model (0=normal; 4=absent uptake). The extent andseverity of ischemia (SDS) was derived from the difference betweensummed stress score (SSS) and summed rest scores (SRS).

Results: In 14 patients with abnormal SPECT (SSS >4), mean SDS wasgreater with imaging agent 1 than with SPECT (9.6±1.8 versus 5.4±0.7,p=0.02). In a matched group of 13 patients with abnormal SPECT, mean SDSwas similar with ⁸²Rb PET and SPECT (4.9±1.4 versus 4.6±1.3, p=0.8). Inpatients with normal SPECT (SSS <4), no differences in SDS were observedwith either imaging agent 1 (n=12) or ⁸²Rb (n=10) PET when compared toSPECT.

Imaging agent 1 PET showed an increase in the amount of ischemiadetected relative to ^(99m)Tc sestamibi SPECT that was not seen whencomparing ⁸²Rb PET to SPECT in a comparable patient group. These resultssuggest that imaging agent 1 show greater improvement in detection ofmyocardial ischemia when PET is compared to SPECT than is associatedwith the use of ⁸²Rb.

Example 20

The following describes multicenter development of normal perfusion andfunction limits for stress and rest cardiac PET. The study includeddevelopment of normal perfusion distribution limits and characterizationof normal cardiac function measured by a cardiac perfusion ¹⁸F basedagent (imaging agent 1).

Methods: Normal limits were established from 15 low likelihood patients(7F/8M) average age 54.7 y, average weight 74.2 kg with treadmillexercise stress/rest datasets (30 datasets in total), recruited in aclinical trial (phase 2) for the ¹⁸F imaging agent 1 perfusion agent,acquired on a Siemens Biograph-64 PET/CT scanner in list mode. Standardreconstruction (2D Attenuation Weighted Ordered Subsets ExpectationMaximization) with voxel size of 2.6×2.6×2.0 (mm) 8-bin gating was usedfor the gated reconstruction. 5-minute reconstructions were consideredobtained approximately 5 min after isotope injection for stress andrest. The Cedars-Sinai QPET PET function and perfusion analysis softwarewas used for all the processing and for the normal perfusion databasecreation. 2 out of 30 scans (6.7%) for gated studies and 1 out of 30 forungated studies (3.3%) required manual intervention in the definition ofthe left ventricle (LV) all other processing was fully automatic.

Results: Left ventricular counts were 33.33±6.44 million counts, range(22.76-44.29) for stress and 7.56±1.86 million counts, range(5.12-11.77) for rest. The stress/rest count ratio was 4.53±0.88(2.88-6.16). Average trans-ischemic dilation (TID) was 0.974±0.124 withupper normal limit of 1.22. QPET relative perfusion normal limits werecreated for stress and rest scans. There was evidence of apical thinningon stress and rest with apical counts at 80/79% respectively. Thevariation of counts in the normal database was between 5-9% in all 17AHA segments. The functional parameters are given in Table 2:

TABLE 2 Functional Parameters from Stress and Rest Scans EDV ESV EF PFRTTPF Stress 96.1 ± 25.2 ml 33.5 ± 14.2 ml 65.9 ± 6.3%  2.12 ± 0.49   205± 51 ms (52.5-74.4%) Rest 91.2 ± 20.0 ml 31.6 ± 12.7 ml 66.5% ± 8.4% 2.39 ± 0.50 162 ± 24.8 ms (50-80%)

Example 21

The following describes results of absolute quantification of rest andstress myocardial blood flow with imaging agent 1 PET in normal andcoronary artery disease patients. Imaging agent 1 is a new myocardialperfusion PET tracer that targets mitochondrial complex 1. In thisstudy, the quantification of rest (R) and stress (S) myocardial bloodflows (MBFs) and coronary flow reserve (CFR) was explored with thistracer in normal and coronary artery disease (CAD) patients.

Methods: Eleven patients (8 with a low likelihood of CAD and 3 with CADand presence of reversible defects) received IV injection of imagingagent 1 at rest and at peak adenosine pharmacological vasodilation.Dynamic PET images were obtained for 10 minutes, beginning with theadministration of the tracer. On reoriented short axis images, regionsof interest were placed on the normal and defect regions of themyocardium and the left ventricular blood pool, from which time activitycurves (TACs) were generated. Patlak analysis was applied to myocardialTAC (˜0.4-1.5 min) using the blood pool TAC as the input function togive the uptake constant (K) in the myocardium. Partial volume andspillover corrections were applied to blood pool and myocardial TACs toensure the intercept of the regression line on the Patlak plot was closeto zero. The first pass extraction fraction for imaging agent 1 inhumans was assumed to be 0.94 (i.e., MBF=K/0.94), equivalent to thatobserved in prior studies (e.g., see Huisman et al., J Nucl Med 2008;49:630-6).

Results: S MBF was similar (p=NS) in LL patients and in the myocardialregions which were supplied by normal coronary arteries in CAD patients(NCA). R MBF, however, was higher (p<0.05) in NCA versus LL, resultingin a lower (p<0.05) CFR in NCA patients. In contrast, S MBF and CFR weresignificantly lower in CAD regions (see Table 3). These findings are inagreement with the published literature using N-13 ammonia PET.

TABLE 3 Rest MBF Stress MBF CFR LL 0.66 ± 0.12 2.36 ± 0.49 3.73 ± 1.24NCA 0.90 ± 0.15 2.38 ± 0.23 2.68 ± 0.32 CAD 0.76 ± 0.13 1.18 ± 0.25 1.58± 0.33

The study data showed that absolute MBF could be quantified at rest andstress in humans using imaging agent 1 myocardial perfusion PET imaging.

Example 22

The following describes an iterative technique for optimizing injectedtracer dosage and acquisition time for ¹⁸F-labeled myocardial perfusiontracer imaging agent 1. Public and staff concerns about radiationexposure necessitate optimization of the dosage acquisition time product(DATP) to obtain the optimal tradeoff between dose, acquisition time,and image noise. An iterative algorithm was developed for determiningoptimal dosage and acquisition time based on a task-limited noise level.

Methods: The mean and standard deviation (SD) were determined from aregion of interest (ROI) of the myocardium to define a ratio: mean/SD(MSD). Using SD and a surrogate for “noise” has its limitations: 1)intrinsic count variability due to partial volume, and tracer uptakeand, 2) non-Poisson nature of reconstructed and post filtered data. Theiterative algorithm was used to fit a model to the limiting MSD. Fromthis, an optimal acquisition time was determined for a target MSD todetect a 5% perfusion defect.

Data Acquisitions: Phantom data simulating patient distributions and a40% septal defect were acquired on a Biograph 64 slice PET/CT scannerusing a 30 minute listmode acquisition. The technique was also tested in18 subjects. Patients received a 2 mCi at rest and ˜2 mCi stress on thefollowing day. A dynamic series for 10, 20, 40, 80, 160, and 320 secondswas acquired 10 minutes post injection. The myocardial ROI was takenfrom a separate 600 second acquisition using >70% of the maximummyocardial voxel limit

Data Analysis: The phantom data converged to theoretical DATP of 9.5 mCi(simulated)*min. In patients, the iterative algorithm converged to asolution in 18 Rest, 9 Ad and 8 Ex. The results are summarized in Table4:

TABLE 4 DATP for detection of 5% defect. 95% time is the limit in which95% of patients would have detection of the 5% defect. 95% ACQ TIME MEANSTDEV (for 1 mCi) REST 2.48 1.25 4.98 EX Stress 1.80 0.57 2.94 AD Stress1.22 0.55 2.32

The iterative technique for solving for optimal dosage acquisition timeproduct converged for phantom and patient studies. Using this result, anoptimal acquisition times for rest, adenosine and exercise stress wasdetermined. Furthermore, it was determined that the algorithm can beused to test alternative filtering, and detection limit and used toextrapolate to the performance of lower sensitivity scanners.

Example 23

The following describes the independence of myocardial functionalparameters (LVEF, EDV and ESV) across a large range of acquisition timesas measured from radiotracer, imaging agent 1. The accurate measurementof functional parameters using myocardial perfusion PET requiresadequate count density. The correlation of functional parameters [leftventricular ejection fraction (LVEF), end-systolic volume (ESV), andend-diastolic volume (EDV)] were examined with acquisition time.

Methods: To analyze the robustness of functional measurements tovariations in count density, a series of low count [1, 3, 5 minuteadenosine (AD), 3, 5 minute rest, 5, 10 minute exercise (EX)] to highcount (15 minute AD, 10 minute rest, 15 minute EX) ECG gated (16 timebin) PET data sets from “listmode” data were produced. LVEF, ESV and EDVwere measured using the QPET analysis program.

Data acquisition: from 23 patients from two study centers. Data for thisstudy was acquired using a same day, rest stress study. Patientsreceived ˜2 mCi at rest and also received a ˜6 mCi “same day” stress (8EX, 13 AD) dosage. Functional values from shorter rebinning times werecompared with the longest acquisition time dataset. Correlations weredetermined using linear regression analysis.

Results: For all acquisition times examined, regression slopes werewithin 10% of unity (with the exception of the 1 minute adenosine, 20%).Correlation coefficients are in Table 5.

TABLE 5 Correlation coefficient between list rebinning and longestacquisition. EDV ESV LVEF 3 min -rest 0.970 0.985 0.985 5 min-rest 0.9950.990 0.985 1 min-AD 0.970 0.975 0.906 10 min-AD 0.997 0.998 0.995 5min-EX 0.990 0.995 0.990 10 min-EX 0.999 0.999 0.999

The high count density present in cardiac imaging agent 1 myocardialperfusion PET images showed robust functional measurements across a widerange of count densities is possible. Modest variations in parametersaffecting count density, such as BMI and variations in dosage, areunlikely to alter functional measurements.

Example 24

The following describes the development of a method for thedetermination of minimum inter-injection interval for a one-dayrest-stress protocol with imaging agent 1 PET myocardial perfusion. Aone-day rest-stress protocol for myocardial perfusion imaging (MPI)needs minimization of wait time between injections (WT) as shorter timesrequire greater stress/rest dosing ratios (DR) and minimum rest dose isdictated by image statistics. A method for determining the dependence ofDR on WT and to identify a WT for acceptable total dose was developed.

Methods: Two-day rest-stress imaging agent 1 PET image data of the heart(5 adenosine (AD) and 5 exercise (EX) stress) from 20 patients withknown reversible defects on Tc-99m MPI were combined to createartificial blended images by adding 16%, 23%, 48% or 100% of rest imageto the stress image. These were paired with rest images, 2-day stressimages and read by 3 blinded readers. Results were recorded by segmentas reader response (RR) (0 to 4) and as quantitative defect severity(QDS) in % decrease.

Results: RR was found to be linearly related to the QDS. In general,decreases greater than 80% of maximum were read as 0, 70% to 80% as 1,60% to 70% as “2,” 50% to 60% as “3” and below 50% as “4.” Analysis ofRR indicated that greater than 1 unit change from the 2-day data wereobserved in reader response in general only for the 48% and 100% blendedimage sets. Therefore 23% was deemed the maximum tolerablerest-to-stress contamination. Using the relationship between rest-stresscontamination and dosing, it was found that, for AD a minimum DR of 2.2was required with a 0.5 hour WT, and for EX a minimum DR of 3.0 wasneeded with a 1-hour WT.

Maximum tolerated rest-to-stress contamination levels were determinedfrom modeled images. The uptake properties of imaging agent 1 withelevated coronary flow made it possible to tolerate a relatively low DRand short WT for AD studies while a longer WT and higher DR is neededfor EX studies.

Example 25

The following describes the design of a 1-day rest-stress PET MPIprotocol that requires selection of doses and imaging times for bothrest and stress phases as well as the interval between rest and stressdoses.

These parameters were determined using three properties of imaging agent1 in myocardial perfusion imaging: 1) the injected dose at rest thatyields a diagnostic quality image for a given acquisition time, 2) themaximum acceptable contribution of the rest dose to the stress image and3) the maximum total injected dose that may be administered based onradiation dose considerations.

The minimum rest dose for a given imaging acquisition time in which thecount-related signal-to-noise did not meaningfully contribute to readererror was determined. This was done by simulating increasing doses usingmultiple rebinnings of patient rest study with increasingly greateramounts of data. This method uses the increasing number of coincidenceevents in sequential rebinnings to create images that model increasingdose and/or acquisition duration. This method is valid for relativelylow concentrations of radioactivity, such as are used here.

When the relationship between the dose and acquisition time are known,the rest dose for Cohort 2 was calculated. After considering the dosingrequired for a range of acquisition times from two minutes up to apractical maximum of 10 minutes, five minutes was selected. Thispermitted an initial dose of 2.9 mCi for the rest acquisition.

To determine the stress dose for a given rest dose, the dosing ratio wasdetermined. To do this, first, the maximum tolerable contribution of therest dose to the-stress image was determined. This was assessed bycreating simulated stress images with a range of rest dose contributionsusing combinations of data from the Study Day 1 rest and Study Day 2stress studies.

The final step of the method is the need to maintain the total dosebelow a limit of 14 mCi, with some additional margin, to limit theradiation dose to 5 rem to the critical organ and 1 rem effective dose(ED) or lower.

Using the maximum rest contribution to the stress image from theanalysis, a range of dosing intervals was considered from a minimum of15 minutes (essentially immediately) to a maximum practical limit of 2hours. Based on this it was possible to select a 30 minute interval foradenosine stress that yielded a corresponding ratio of the stress doseto the rest dose of 2.0.

For exercise stress, a combination of a longer dosing interval andgreater dose ratio was needed due to the lower net myocardial uptake ofradioactivity with exercise. Thus, a dosing interval of 60 minutes waschosen which corresponded to a dose ratio of 3.0. The rest acquisitiontime was increased to 7 minutes and the rest dose reduced to 1.7 mCi toallow for the greater required stress/rest dose ratio while stillmaintaining the total comfortably within the 14 mCi limit.

In order to allow some range of dosing and to avoid variations in dosingthat might jeopardize the integrity of the study, the dose and doseratio values above were set as the lower limits of 15% to 20% ranges foreach variable and the acquisition times increased to a minimum of 15minutes for all acquisitions to account for the possibility of lowersensitivity of the 2D PET scanners. The data acquisition was broken intosections so that images derived from shorter acquisition times may beobtained from the same data as necessary. This resulted in the finalspecified dosing of 2.9 mCi to 3.4 mCi rest with a stress dose of 2.0 to2.4 times the rest dose for adenosine stress. For exercise stress, thefinal doses were set at 1.7 mCi to 2.0 mCi for rest with a stress dose3.0 to 3.6 times the rest dose. These doses are intended to reflect theactual net injected radioactivity so that additional radioactivity isrequired in the syringe prior to injection to compensate for losses dueto adsorption and the dead volume of the syringe.

Example 26

The following describes human safety, dosimetry, biodistribution, andrest-stress myocardial imaging characteristics of ¹⁸F-labeled imagingagent 1 myocardial perfusion PET tracer. ¹⁸F-labeled imaging agent 1 isa novel myocardial perfusion imaging PET tracer that targetsmitochondrial complex 1. Studies of human safety, dosimetry,biodistribution, and myocardial imaging characteristics of this tracerwere evaluated.

Methods: 25 normal subjects were enrolled in 2 studies: 13 received 222MBq I.V. At rest (R) only and 12 more subjects received 94 MBq at R and,on a second day, 124 MBq at peak adenosine stress (Adeno, n=6) or atpeak treadmill exercise (Ex, n=6). Physical exam, laboratory, vitalsigns, ECG, and EEG were monitored pre- and post-injection. Myocardial(Myo), liver, blood pool and lung Standardized Uptake Values (SUV) weredetermined from sequential PET images over time. Mean dose for variousorgans and mean effective dose (ED in mSv/MBq) were estimated.

Results: There were no adverse events related to the tracer. The tophighest-dose organs were kidneys at R and heart with Adeno and Ex. EDwas 0.019 at R and with Adeno and 0.015 with Ex. Myo SUV's remained highduring imaging. Ex myo SUV was lower with Ex due to higher skeletalmuscle uptake. Ex myo SUV was lower with Ex due to higher skeletalmuscle uptake. Myo/liver was highest with Ex, followed by Adeno and R(see Table 6). Myo/blood and Myo/lung were high and rapidly improvedwith time.

TABLE 6 10 mins 30 mins 60 mins 90 mins 149 mins Rest Myo SUV 3.9 ± 0.94.2 ± 1.1 4.5 ± 1.2 4.3 ± 1.3 4.1 ± 1.4 Rest Myo/liver 1.0 ± 0.3 0.9 ±0.2 1.1 ± 0.2 1.4 ± 0.2 2.1 ± 0.3 Adeno Myo SUV 10.5 ± 1.5  10.8 ± 2.1 10.3 ± 2.1  9.6 ± 2.1 8.4 ± 2.1 Adeno Myo/liver 1.9 ± 0.6 2.0 ± 0.5 2.2± 0.5 2.6 ± 0.5 3.8 ± 1.0 Exercise Myo SUV 6.2 ± 2.1 5.5 ± 1.0 5.1 ± 0.94.9 ± 0.9 4.5 ± 0.8 Exercise Myo/liver 28.0 ± 33.6 5.6 ± 1.0 5.6 ± 1.35.8 ± 1.5 5.5 ± 1.5

Example 27

Studies were performed in subjects to determine dosing protocols forimaging agent 1 under various conditions. Determining dosing protocolsincluded assessing parameters such as mCi of imaging agent 1 injected inthe body of the subject; mCi of imaging agent 1 injected from thesyringe; acquisition time of images after injection; delay between restand stress studies, etc. Parameters varied for rest and stress, forexample, the injected dose (in the body) for exercise stress was atleast three times the injected dose (in the body) at rest. In addition,the injected dose (in the body) for pharmacological stress was at leasttwo times the injected does (in the body) at rest. Results are shown inTable 7.

TABLE 7 Imaging agent 1 doses, acquisition times and dosing delay forexercise and pharmacologic stress. Injected Dose Injected DoseAcquisition Delay between in the Body in the Syringe Time Studies StressTest Study (mCi) (mCi) (min) (min) Exercise Rest 1.7-2.0 2.5-3.0 10 60Stress 8.6 to 9.0 9.0-9.5 Pharmacologic Rest to Minimum Stress ratio x3injected Rest dose Rest 2.4-2.9 2.5-3.0 10 30 Stress 5.7-6.2 6.0-6.5Rest to Minimum Stress ratio x2 injected Rest dose

Various parameters have been determined for dosing imaging agent 1 inhuman subjects, including injected does, delay between studies, rationof rest to stress dosing, and the amount in the syringe compared to theamount injected from the syringe.

Example 28

The following provides results obtained from a study regarding asingle-dose dosimetry, biodistribution, and safety trial of imagingagent 1 in healthy subjects. Whole body PET image data for the 12healthy volunteers were obtained using imaging agent 1 at approximately10 minutes, 30 minutes, 50 minutes, 2 hours, 2.5 hours, 3.83 hours, and4.5 hours post injection. Image data were attenuation corrected at theimaging site, and were quantified based on the Medical InternalRadiation Dose (MIRD) 16 methodology by Dosimetry Analysis Laboratory,CDE Dosimetry Services (CDE) to determine kinetic data in all organsshowing significant uptake of activity. Dosimetry estimates were createdvia kinetic modeling of the quantified image data to determine residencetimes, and the standard MIRD methodology. These estimates weredetermined using 3 assumptions regarding urinary bladder voidingintervals (2.0, 3.5, and 4.8 hr). Kinetic data, residence times, and thedosimetry estimates are reported for individuals, and as summarystatistics.

Terminology: Effective Dose (ED): Developed by the ICRP for occupationalradiation protection, the ED enables the comparison of radiationdetriment from a uniform external dose and a non-uniform internal dose.The risk for a 1 rem ED determined for an non-uniform internal dose isequal to the risk from a 1 rem uniform external exposure (total bodydose). As defined in ICRP publication 60 [ICRP-60 1991].

Effective Dose Equivalent (EDE): Developed by the ICRP for occupationalradiation protection, the EDE enables the comparison of radiationdetriment from a uniform external dose and a non-uniform internal dose.The risk for a 1 rem EDE determined for an non-uniform internal dose isequal to the risk from a 1 rem uniform external exposure (total bodydose). As defined in ICRP publication 30 [ICRP-30 1981].

MIRD Methodology: The methodology developed by the Medical InternalRadiation Dose Committee for the determination of radiation absorbeddose. This methodology included the use of radiation transport factors(S-values), and bio-kinetic parameters (residence times). As defined inthe MIRD Primer, Society of Nuclear Medicine, 1991.% CV is Coefficient of variation(Ratio of the standard deviation to themean times 100).

Percent Injected Dose vs. Time from Whole Body Images. Percent injectedactivity as a function of time was determined for brain, heart wall,kidneys, liver, lungs, red marrow (lumbar region), salivary glands,spleen, stomach wall, thyroid, and urinary bladder. On average, theorgan that showed the largest peak uptake was the liver withapproximately 19.1% of the injected activity (data not shown). The nextlargest peak uptake occurred in the kidneys with approximately 9.4% ofthe injected activity (data not shown).

Dosimetry Estimates. On average, for the urinary bladder voidinginterval of 3.5 hours the organ receiving the largest absorbed dose wasthe kidneys at 0.24 rem/mCi (0.066 mSv/MBq) and the heart wall at 0.18rem/mCi (0.048 mSv/MBq). The mean ED (effective dose) was 0.071 rem/mCi(0.019 mSv/MBq). Table 8 shows the absorbed dose estimates (rem/mCi).The mean adsorbed dose for the listed organs is found in column one ofTable 8.

TABLE 8 Mean % CV Min Max Adrenals 5.8E−02 7% 4.9E−02 6.4E−02 Brain9.4E−02 25%  5.7E−02 1.3E−01 Breasts 3.2E−02 8% 2.8E−02 3.5E−02Gallbladder Wall 6.4E−02 8% 5.4E−02 7.1E−02 LLI Wall 4.3E−02 8% 3.7E−024.8E−02 Small Intestine 4.7E−02 8% 4.0E−02 5.2E−02 Stomach Wall 1.5E−0126%  9.0E−02 2.3E−01 ULI Wall 4.7E−02 7% 4.1E−02 5.2E−02 Heart Wall1.8E−01 17%  1.2E−01 2.4E−01 Kidneys 2.4E−01 22%  1.6E−01 3.5E−01 Liver1.5E−01 19%  1.0E−01 1.9E−01 Lungs 4.2E−02 7% 3.6E−02 4.6E−02 Muscle3.8E−02 8% 3.2E−02 4.1E−02 Ovaries 4.5E−02 8% 3.9E−02 5.0E−02 Pancreas5.9E−02 8% 4.8E−02 6.7E−02 Red Marrow 6.0E−02 11%  4.7E−02 6.9E−02Osteogenic Cells 6.9E−02 8% 5.7E−02 7.8E−02 Salivary 1.3E−01 38% 8.6E−02 2.5E−01 Skin 2.9E−02 8% 2.5E−02 3.2E−02 Spleen 6.0E−02 21% 4.0E−02 7.6E−02 Testes 3.4E−02 9% 3.0E−02 3.8E−02 Thymus 4.1E−02 8%3.5E−02 4.5E−02 Thyroid 1.2E−01 30%  7.1E−02 1.8E−01 Urinary BladderWall 8.4E−02 18%  6.5E−02 1.1E−01 Uterus 4.6E−02 8% 4.0E−02 5.1E−02Total Body 4.5E−02 7% 3.7E−02 4.9E−02 EDE 8.0E−02 11%  6.3E−02 9.1E−02ED 7.1E−02 12%  5.5E−02 8.9E−02

Example 29

Results related to a human study of imaging agent 1, a novel ¹⁸F-labeledtracer for myocardial perfusion PET imaging; dosimetry, biodistribution,safety, and imaging characteristics after a single injection at rest aredescribed.

Methods: Study population. Healthy adults (as determined by medicalhistory, physical examination, vital signs, ECG, EEG, neurologicalexamination, and clinical laboratory testing), ages 18-40 years,participated in the study. In order to be enrolled, subjects had to meetall protocol-specified inclusion criteria and none of the exclusioncriteria.

Study design. This was a non-randomized, open-label, single-dose study.A total of 13 healthy adult subjects were enrolled and administered asingle dose of imaging agent 1 at a single study center in the UnitedStates. Subjects were screened within 14 days prior to enrollment toconfirm subject eligibility, and began baseline assessments at the studycenter the day before study drug administration. Subjects remained atthe study center until completion of the Study Day 2 safety assessments(24±8 hours post-dose). A telephone call was made to study subjects 48±8hours post-dose for adverse event (AE) monitoring. All subjects returnedto the study center approximately one week (5-7 days) post-dose for afollow-up safety visit, and were contacted by telephone approximately14-17 days post-dose for final serious AE monitoring.

Determination of dose and method of administration. The 8 mCi targetdose was selected to provide adequate count statistics and was projectedto be well below the maximum acceptable radiation exposure based onpreclinical data. These data demonstrated that the maximum dose ofimaging agent 1 that may be administered to a human without exceeding 50mSv (5 rem) to the target was 742 MBq (20.0 mCi) and the injected dosethat yielded an effective dose (ED) of ≦10 mSv (1 rem) was 666 MBq (18.0mCi) (Stabin, M G, Sparks, R B, et al, OLINDA/EXM: the second-generationpersonal computer software for internal dose assessment in nuclearmedicine.” J Nucl Med 2005 46(6):1023-7).

On day 1, each subject received a 1-3 mL intravenous bolus injection ofimaging agent 1 in a sterile solution of <5% ethanol containing <50mg/mL sodium ascorbate in water, calculated to deliver approximately thetarget dose of imaging agent 1 at the time of injection. The dose wasadministered in less than 10 seconds, followed immediately by a 3-5 mLsaline flush.

The net injected dose was calculated by subtracting the decay-correctedradioactivity in the syringe and injection tubing after injection fromthe assayed and decay-corrected radioactivity in the syringe prior toinjection.

PET imaging protocol. Whole-body PET imaging from head to mid thigh wasperformed at protocol-specified time-windows.

Dosimetry analyses. Estimates of radiation dosimetry for the standardorgans of the adult male and female models and for the, salivary glandsas well as the effective dose equivalent (EDE) (International Commissionon Radiological Protection (ICRP), Recommendations of the InternationalCommission on Radiological Protection, Publication 26. Ann ICRP. 1977;1(3)) and the effective dose (ED) (International Commission onRadiological Protection (ICRP), 1990 Recommendations of theInternational Commission on Radiological Protection, 60. Ann ICRP. 1990;21(1-3)) were determined using the OLINDA/EXM software (Stabin, M G,Sparks, R B, et al, OLINDA/EXM: the second-generation personal computersoftware for internal dose assessment in nuclear medicine.” J Nucl Med2005 46(6):1023-7) Assessment of radiation dosimetry was based on theMIRD method, with data derived from imaging studies, using methodsconsistent with MIRD Pamphlet no. 16 (Siegel J A, Thomas S R, Stubbs JB, et al. MIRD pamphlet no. 16: Techniques for quantitativeradiopharmaceutical biodistribution data acquisition and analysis foruse in human radiation dose estimates. J Nucl Med. 1999 February;40(2):37S-61S).

The attenuation corrected transverse image data slice planes werecombined into a single three-dimensional image matrix for each subjectand each time point using custom software. These images were thendivided into 6 image sets (“anterior”, “posterior”, “salivary”,“thyroid”, “source”, and “full”) of combined coronal plane image datafor each subject at each time point, grouping organs with similaranterior to posterior depths. This was done to optimize the ROIcreation, and minimize background contribution to the organs containedin each combined coronal plane image. The “anterior” images containedstomach wall, heart wall, and urinary bladder. The “posterior” imagescontained kidneys, lumbar spine, and spleen (when visible). The“salivary” images contained the salivary glands (parotid andsubmandibular). The “thyroid” images contained the thyroid. The “full”images combined all of the coronal image planes that contained subjectimage data and was used for quantification of the brain and liver. The“source” images contained the calibration source.

Regions of interest were drawn around all organs that showed uptakeabove background using custom software developed and validated for thispurpose. Absolute radioactivity was determined by normalizing ROI sumsby a calibration factor derived from the calibration source. Regioncounts were also adjusted for activity containing underlying andoverlying tissue that was not part of the organ or tissue beingquantified by utilization of background regions of interest. Total bodyregion counts were also corrected for off body background counts.Appropriate normalization of region sizes for organ and adjacent regionswere made. Unobstructed regions of organs with significant overlap fromother activity containing organs were also employed where necessary. Inorder to estimate the activity in the lower legs (which were notimaged), a region of interest on the upper thigh was utilized.Activities were also normalized where necessary to account for 100% ofthe injected activity, and to insure conservative (slightover-estimates) determination of absorbed dose. Where urinary excretiondata were available beyond the end of the imaging regimen, these datawere used to determine whole body retention.

Kinetic data for brain, heart wall, kidneys, liver, red marrow (lumbarspine regions were utilized), salivary glands, spleen, stomach wall,thyroid, and urinary bladder for the subjects in the study weredetermined using image quantification methodology. Absolute activity wasconverted to fractional dose by dividing by the total activityadministered. Organ and tissue data were fit using non-linearleast-squares regression with sums of exponentials of the form shown inEquation 1, where f and X, are the model parameters that are determinedin the fitting process, F_(ij)(t) is the fraction of the total injectedactivity, t is the time post injection, i is the i^(th) ROI, j is thej^(th) subject, and k is the k^(th) exponential term. Between one andfour exponential terms were employed, as appropriate.

$\begin{matrix}{{F_{ij}(t)} = {\sum\limits_{k}{f_{ijk}{{\mathbb{e}}^{{- \lambda_{ijk}}t_{j}}.}}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

The regression was performed using custom software that determinesinitial parameter values based on the temporal variation of the kineticdata, and the use of pre-tabulated estimates for various time activityscenarios as selected by the user. Once these data were fit, residencetimes were determined by integration of these empirically determinedfunctions (sums of exponentials) from time equal zero to infinity,taking into account physical decay. Remainder of Body residence timeswere determined by subtraction of appropriate organ residence times fromwhole body residence times. Urinary bladder residence times weredetermined using the parameters determined by fitting the whole bodyactivity data with a urinary bladder model as implemented in theOLINDA/EXM software with 3.5 hour bladder voiding interval. Red marrowresidence time was determined based on a region of interest drawn on aportion of the lumbar spine. The lumbar spine was assumed to contain16.1% (International Commission on Radiological Protection (ICRP)Publication 23, Report of the Task Group on Reference Man. PergamonPress. 1975, page 125) of the total red marrow.

Organ/Tissue Dosimetry Estimates. Absorbed dose estimates for all targetorgans were determined using the OLINDA/EXM software using the adult“male” model. The resulting absorbed dose estimates were scaled based onthe total body mass of the individual subjects relative to that of theradiation transport phantom. Salivary gland dosimetry was determined byusing a conservative estimate of the S-value for salivary glands basedon the reference man total mass of the parotid and submaxilary salivaryglands (International Commission on Radiological Protection (ICRP)Publication 23, Report of the Task Group on Reference Man. PergamonPress. 1975, page 125) and assuming a spherical shape. S-Values forspheres were produced by the OLINDA/EXM software, and were linearlyscaled based on the relative total body mass of reference man to that ofsubject. These S-values were then multiplied by the residence times toproduce final salivary gland dose estimates.

Statistical Analyses. All statistical analyses and all summary tablesand listings were prepared using SAS® release 9.1.3 (SAS Institute,Inc., Cary, N.C.). Standard descriptive summaries included the N, mean,median, standard deviation (SD) and/or co-efficient of variation (% CV),minimum and maximum for continuous variables, and the number and percentfor categorical variables.

Results: Patient demography. Of the 26 subjects who were screened, 13subjects (12 males and one female) were administered imaging agent 1,and completed all safety evaluations. The mean age was 23.4 years(range: 19-34 years) and the mean BMI was 23.4 (range: 20-26). Onepatient was not included in the analyses of dosimetry, biodistributionand radiokinetics, due to the inability to confirm the dose calibratorassay data for the standards preparation.

Radiation dosimetry. The intravenous bolus injection was calculated todeliver no more than 8 mCi of ¹⁸F at the time of injection. The mean(SD) final decay-corrected dose was 6 (0.6) mCi of ¹⁸F, with a range of4.6 to 6.6 mCi (170 to 244 MBq). The difference between the target doseand the final dose was due to the retention of imaging agent 1 in thesyringe.

The absorbed dose summary statistics are presented in Table 9 (mSv/MBq).The organ receiving the largest mean absorbed dose was the kidneys at0.066 mSv/MBq (0.24 rem/mCi), followed by the heart wall at 0.048mSv/MBq (0.18 rem/mCi). The mean ED was 0.019 mSv/MBq (0.072 rem/mCi).

TABLE 9 Absorbed Dose Estimates (mSv/MBq), N = 12, Void Interval = 3.5hours. Mean % CV Min Max Adrenals 1.6E ^(a)−02   7% 1.3E−02 1.7E−02Brain 2.5E−02 25%  1.5E−02 3.6E−02 Breasts 8.8E−03 8% 7.5E−03 9.6E−03Gallbladder Wall 1.7E−02 8% 1.5E−02 1.9E−02 LLI Wall 1.2E−02 8% 1.0E−021.3E−02 Small Intestine 1.3E−02 8% 1.1E−02 1.4E−02 Stomach Wall 4.0E−0226%  2.4E−02 6.2E−02 ULI Wall 1.3E−02 7% 1.1E−02 1.4E−02 Heart Wall4.8E−02 17%  3.4E−02 6.4E−02 Kidneys 6.6E−02 22%  4.4.E−02  9.5E−02Liver 3.9E−02 19%  2.7E−02 5.2E−02 Lungs 1.1E−02 7% 9.7E−03 1.2E−02Muscle 1.0E−02 8% 8.7E−03 1.3E−02 Ovaries 1.2E−02 8% 1.1E−02 1.3E−02Pancreas 1.6E−02 8% 1.3E−02 1.8E−02 Red Marrow 1.6E−02 11%  1.3E−021.9E−02 Osteogenic Cells 1.9E−02 8% 1.6E−02 2.1E−02 Salivary 3.5E−0238%  2.3E−02 6.8E−02 Skin 7.9E−03 8% 6.8E−03 8.7E−03 Spleen 1.6E−02 21% 1.1E−02 2.1E−02 Testes 9.2E−03 9% 8.1E−03 1.0E−02 Thymus 1.1E−02 8%9.6E−03 1.2E−02 Thyroid 3.2E−02 30%  1.9E−02 4.9E−02 Urinary BladderWall 2.3E−02 18%  1.7E−02 3.0E−02 Uterus 1.2E−02 8% 1.1E−02 1.4E−02Total Body 1.2E−02 7% 1.0E−02 1.3E−02 EDE 2.2E−02 11%  1.7E−02 2.5E−02ED 1.9E−02 12%  1.5E−02 2.4E−02 ^(a) “E” followed by a “−” is theexponent multiple of 3 convention for decimal presentation.

Whole-organ biodistribution. The biodistribution of imaging agent 1,calculated as the whole-organ percent injected radioactivity as afunction of time, was determined for brain, heart wall, kidneys, liver,lungs, red marrow (lumbar region), salivary glands, spleen, stomachwall, thyroid, and urinary bladder (Table 10 and FIG. 11). FIG. 11 showswhole body coronal images through the body at the level of themyocardium from a representative subject at different time points afteradministration of imaging agent 1. Images have been corrected for ¹⁸Fdecay. It can be seen that the heart exhibits high and sustainedretention of ¹⁸F from the earliest images through approximately 5 hoursafter injection. The liver also appears, generally exhibiting anintensity similar to that of the heart, peaking between 10 and 30minutes after injection and clearing by approximately 2 hours. The organthat showed the largest mean peak uptake was the liver withapproximately 19.1% of the injected activity. The next largest mean peakuptake occurred in the kidneys with approximately 9.4% of the injectedactivity, followed by the brain with approximately 8.3% of the injectedactivity. Data from subjects in the study were used to determine theurinary excretion rate for each subject and the residence time forradioactivity in the bladder using a standard model, with a theoreticalfixed voiding interval of 3.5 hours post-dose. The largest meanresidence times were for remainder tissues (1.8 hours), liver (0.28hours), and brain (0.14 hours). Summary residence time statistics arepresented in Table 11.

TABLE 10 Mean Percent (%) Administered Dose versus Time (HoursPost-dose) N = 12, ¹⁸F, Decay Corrected. 0.17 hr ^(a) 0.50 hr 0.83 hr2.0 hr 2.5 hr 3.83 hr 4.5 hr Whole Body 100.0% 99.8% 99.7% 98.2%  98.1% 96.8%  96.9%  Brain 8.3% 7.9% 7.3% 4.7% 4.1% 3.2% 2.9% GI Stomach Wall2.5% 2.4% 2.2% 0.7% 0.7% 0.6% 0.6% Heart Wall 3.1% 3.2% 3.4% 2.4% 2.5%2.1% 2.1% Kidneys 9.4% 6.5% 4.9% 1.6% 1.6% 1.2% 1.1% Liver 19.1% 18.0%16.4% 7.5% 7.0% 4.5% 4.7% Marrow (lumbar) 0.3% 0.3% 0.3% NA NA NA NASalivary 0.6% 0.7% 0.6% 0.5% 0.5% 0.4% 0.4% Spleen 0.9% 0.6% 0.4% 0.3%0.3% 0.3% NA Thyroid 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% Urinary Bladder0.3% 0.2% 0.3% 0.9% 1.1% 1.4% 1.7% ^(a) Nominal times in hours post-dose(beginning of time window) NA = not available

TABLE 11 Residence Times (Hours) Summary Statistics (N = 12, VoidInterval = 3.5 Hours). Mean % CV Min Max Brain 1.38E−01 2.69E−018.68E−02 2.08E−01 GI Stomach Wall 3.30E−02 4.46E−01 1.53E−02 6.11E−02Heart Wall 7.28E−02 1.83E−01 4.82E−02 1.01E−01 Kidneys 9.52E−02 2.35E−016.10E−02 1.43E−01 Liver 2.77E−01 2.22E−01 1.83E−01 3.94E−01 Red Marrow8.86E−02 2.10E−01 6.72E−02 1.17E−01 Salivary 1.48E−02 3.24E−01 9.68E−032.63E−02 Spleen 1.01E−02 1.87E−01 7.26E−03 1.25E−02 Thyroid 3.31E−033.13E−01 1.72E−03 5.06E−03 Urinary Bladder 2.65E−02 3.35E−01 1.40E−024.50E−02 Remainder of Body 1.84E+00 7.86E−02 1.65E+00 2.08E+00 ^(a) “E”followed by a “−” is the exponent multiple of 3 convention for decimalpresentation

Early elimination of ¹⁸F in urine. Urine collected pre-dose (Baseline),and all voids up to 8 hours post-dose were collected and assayed for¹⁸F. However, as in blood collection, the urine collection terminatednear the 7-hour minimum specified in the protocol. Mean urinaryexcretion over the approximate 7-hour void interval was 4.83% ID with a% CV of 64.7 and a range of 0.64% ID to 12.41% ID. This finding is inreasonable agreement with cumulative urine excretion of 5% as measuredwith PET imaging.

Discussion: The critical organ for imaging agent 1 was the kidneys, witha mean estimated dose of 0.066 mSv/MBq (0.24 rem/mCi) The maximuminjected dose of the compound that may be administered without exceeding50 mSv to the critical organ is therefore 770 MBq. This is somewhathigher than the 185 MBq to 370 mBq recommended in the widely-usedguidance by the Center for Drug Evaluation and Research (CDER) thatdescribes recommended package insert wording for facilities applying tomanufacture [¹⁸F]-FDG) (PET Drug Applications—Content and Format forNDAs and ANDAs: Fludeoxyglucose F 18 Injection, Ammonia N 13 Injection,Sodium Fluoride F 18 Injection, Attachment II, Sample Formats; Labelingfor Ammonia N 13 Injection, Fludeoxyglucose F 18 Injection and SodiumFluoride F 18 Injection, Attachment II (CDER 2000)). This behavior is aresult of the very rapid urinary excretion of a large fraction of[¹⁸F]-FDG shortly after administration, resulting in a substantiallyhigher exposure to the urinary bladder for that compound compared withthat of imaging agent 1. The ED due to imaging agent 1 (0.019 mSv/MBq),is the same as the ED of [¹⁸F]-FDG (International Commission onRadiological Protection (ICRP), Radiation Dose to Patients fromRadiopharmaceuticals, Addendum 2 to ICRP Publication 53, Publication 80,Ann ICRP. 1999; 28(3)). It can therefore be concluded that the radiationdose from imaging agent 1 is comparable to or less than that due to[¹⁸F]-FDG.

Since the mean estimated effective dose (ED) for imaging agent 1 is0.019 mSv/MBq (0.072 rem/mCi), the maximum injected dose that may beadministered without exceeding 10 mSv ED is therefore 521 MBq.

The radiation dose estimates from this study are consistent with thosederived from non-human primates (Lazewatsky J, Azure M, Guaraldi M etal. Dosimetry of BMS747158, a novel 18F labeled tracer for myocardialperfusion imaging, in nonhuman primates at rest. J Nucl Med. 2009;49(Supplement 1):15p.) and the high and sustained retention of imagingagent 1 in the heart is consistent with data in both non-human primatesand in other species (Yu M, Guaraldi M T, Mistry M, Kagan M, McDonald JL, Drew K, Radeke H, Purohit A, Azure M, Casebier D S, Robinson S P.BMS-747158-02: a Novel PET Myocardial Perfusion Imaging Agent. JournalNuclear Cardiology 2007 November-December; 14(6):789-98). Although thecritical organ in the primate-derived estimates was seen to be the heartwall, the estimated human radiation dose for the heart wall in thatstudy was 0.067 mSv/MBq, which is very similar to the critical organvalue of 0.066 mSv/MBq seen for the kidneys in this study. The doses toboth organs were among the highest in both the non-humanprimates-derived results and in the current study and are within twostandard deviations of one another.

Imaging agent 1 was well-tolerated and no clinically significant safetyconcerns were raised. Changes from baseline in vital signs, laboratoryvalues (hematology, coagulation, clinical chemistry and urinalysis),ECGs, and EEGs were not clinically significant. Potential cardiotoxicity(signaled through coagulation studies and changes in Troponin-T levels)were not exhibited. Physical and neurological examinations did notreveal any pre-dose or post-dose abnormalities. The DMC did not raisesafety concerns following periodic reviews of the safety data.

The results obtained in this study demonstrated that imaging agent 1appeared to be safe and was well tolerated and exhibited a substantialand sustained retention in myocardium. The critical organ after restinginjection of imaging agent 1 was determined to be the kidneys with 0.066mSv/MBq. Based on the observed mean ED, the maximum injected dose thatmay be administered without exceeding 1 rem ED is 14 mCi (521 MBq). TheED from imaging agent 1 is the same as that of [¹⁸F]-FDG, while thecritical organ (kidney) dose of imaging agent 1 is significantly lessthan the critical organ (urinary bladder) dose of [¹⁸F]-FDG.

Example 30

The following example describes studies relating to cardiac imaging andsafety evaluation of imaging agent 1, a novel PET myocardial perfusionimaging agent, in chronic myocardial compromised rabbits.

Imaging agent 1 is an ¹⁸F labeled imaging agent for myocardial perfusionimaging (MPI) with positron emission tomography (PET) (Yu M, Guaraldi MT, Mistry M, Kagan M, McDonald J L, Drew K et al.: a novel PETmyocardial perfusion imaging agent. J Nucl Cardiol 2007; 14:789-98).Cardiac imaging with this agent shows clear myocardium andidentification of acute myocardial ischemia and tissue necrosis inanimal models of acute coronary ligation and ischemia reperfusion injury(Yu M, Guaraldi M T, Mistry M, Kagan M, McDonald J L, Drew K et al.: anovel PET myocardial perfusion imaging agent. J Nucl Cardiol 2007;14:789-98; Nekolla S G, Reder S, Higuchi T, Dzewas G, Poethko T, PreisslA et al. Assessment of Imaging Properties of a New F-18 Labelled FlowTracer in a Pig Model. J Am Coll Cardiol 2008; 51:A170; and Maddahi J,Schiepers C, Czernin J, Huang H, Schelbert H, Wijatyk A et al. Firsthuman study of BMS747158, a novel F-18 labeled tracer for myocardialperfusion imaging. J Nucl Med 2008; 49:70P). In model systems imagingagent 1 has demonstrated superior characteristics over the currentlyavailable MPI agents. In comparison with single photon emission computertomography (SPECT) based agents (^(99m)Tc-Sestamibi and ²⁰¹Thalium),Imaging agent 1 has the advantage of PET technology with accurateattenuation correction and quantification of myocardial perfusion inabsolute terms. Furthermore, imaging agent 1 heart uptake correlatesbetter with myocardial perfusion at a large range of flow rates invitroland at rest and stress conditions in-vivo (Nekolla S G, Reder S, SarasteA, Higuchi T, Dzewas G, Preissel A et al. Evaluation of the novelmyocardial perfusion positron-emission tomography tracer18F-BMS-747158-02: comparison to 13N-ammonia and validation withmicrospheres in a pig model. Circulation 2009; 119:2333-42). Incomparison with current PET agents, like ¹³N-Ammonia and ⁸²Rubidium, thelong half-life (110 minutes) of ¹⁸F enables imaging agent 1 to beradio-synthesized and supplied centrally. It also provides theopportunity for imaging under excise stress, in addition topharmacological stress.

Safety and radio-dosimetry studies in multiple normal species showimaging agent 1 has acceptable safety margin for clinical development(Mistry M, Onthank D, Green J, Cicio S, Casebier D, Robinson S et al.Toxicological Evaluation of BMS-747158, a PET Myocardial PerfusionImaging Agent. The Toxicologist 2008; 102:476; and Lazewatsky J, AzureM, Guaraldi M, Kagan M, MacDonald J, Yu M et al. Dosimetry of BMS747158,a novel 18F labeled tracer for myocardial perfusion imaging, in nonhumanprimates at rest. J Nucl Med 2009; 49:15p). The critical organ forradiation is the heart and the radiation doses were comparable with thecommercial available agent ¹⁸F-fluorodeoxyglucose (Lazewatsky J, AzureM, Guaraldi M, Kagan M, MacDonald J, Yu M et al. Dosimetry of BMS747158,a novel 18F labeled tracer for myocardial perfusion imaging, in nonhumanprimates at rest. J Nucl Med 2009; 49:15p).

Methods: Rabbit Model of Myocardial Infarction. Male New Zealand rabbits(body weight 2.5-3.5 kg) were purchased from Harlan (Oakwood, Mich.) andmaintained in the AAALAC-accredited Animal Care Facility at LantheusMedical Imaging. The study protocol was approved by the InstitutionalAnimal Care and Use Committee. The procedure of developing a rabbitmodel of myocardial infarction (MI) was similar to the method describedpreviously (Fujita M, Morimoto Y, Ishihara M, Shimizu M, Takase B,Maehara T et al. A new rabbit model of myocardial infarction withoutendotracheal intubation. J Surg Res 2004; 116:124-8). Briefly, therabbit was anesthetized with ketamine (40 mg/Kg, im) and xylazine (9mg/Kg, im) and placed in a supine position. The surgery was performedunder aseptic conditions. A mid-sternotomy was performed carefully toavoid injury of parietal pleura. The pericardial sac was exposed andincised. The left ventricular anterior and lateral wall was revealed anda major branch of the left coronary artery was ligated. Success of theligation was verified by the color change to pale in the affected areaof the left ventricular wall. The chest was then closed and the animalallowed to recover. Four weeks after the surgery, the rabbit was usedfor the imaging and cardiovascular evaluation study.

Imaging and Cardiovascular Evaluation. PET images and cardiovascularparameters were evaluated in both normal and MI rabbits. Prior toimaging, the rabbit was anesthetized with ketamine (25 mg/Kg, im) andxylazine (5 mg/Kg, im) and the marginal ear vein was catheterized forimaging agent 1 injection. The right femoral artery was isolated andcanulated with a Millar catheter (SPC340, Millar Instruments, Houston,Tex.) for arterial pressure measurement. Then the animal was positionedin a microPET camera (Focus220, CTI Molecular Imaging, Inc. Knoxville,Tenn.) for cardiac imaging. The Millar catheter was connected to acomputer driven data acquisition system (MP35, BIOPAC Systems, Goleta,Calif.) for recording of mean arterial pressure (MAP), and systolic anddiastolic arterial pressure (SAP and DAP). In addition,electrocardiogram (ECG) was also recorded with 3 non-invasive limb leadsin lead II configuration using the BIOPAC system. Heart rate (HR) and QTinterval were derived from ECG recording. After a stabilization period,cardiovascular parameters: MAP, SBP, DBP and ECG, were recorded 5minutes before imaging agent 1 intravenous injection (˜1.5 mCi) and therecording continued for additional 20 minutes post-injection. The rabbitwas imaged for 30 minutes.

Image Reconstruction and Analysis. After the acquisition, images werereconstructed in a matrix of 256×256 pixels with 95 transverse slicesusing the OSEM2D algorithm and decay corrected (microPET Manager andASIPro, CTI Molecular Imaging, Inc. Knoxville, Tenn.). The pixel sizewas 0.47 mm and the slice thickness was 0.80 mm. The images werereoriented regarding cardiac axis and serial tomographic cardiac imageframes were then generated for a 10-minute period from 20 to 30 minutes.Polar map images were then generated from reconstructed cardiacshort-axis image using QPS 2008 software (Cedars-Sinai Medical Center,Los Angeles, Calif.).

Radiopharmaceutical Agent. The chemical structure and radiosynthesis ofimaging agent 1 have been described previously (Yu M, Guaraldi M T,Mistry M, Kagan M, McDonald J L, Drew K et al.: a novel PET myocardialperfusion imaging agent. J Nucl Cardiol 2007; 14:789-98; and Purohit A,Radeke H, Azure M, Hanson K, Benetti R, Su F et al. Synthesis andbiological evaluation of pyridazinone analogues as potential cardiacpositron emission tomography tracers. J Med Chem 2008; 51:2954-70). Theradiochemical purity used in this study was 99.1-99.9%, and the specificactivity was 3265-7016 Ci/mmol. The agent was prepared in 5% ethanol(v/v) and 50 mg/ml ascorbic acid in water following the clinicalprotocol.

Data Analysis. Data are expressed as mean±SD and unpaired student t-test(assuming unequal variances) was used for comparison of baseline valuesbetween control and MI rabbits. p<0.05 was considered statisticallysignificant. At each timepoint (before and 1-, 5-, 10- and 20-minuteafter imaging agent 1 injection), MAP, SAP and DAP measuredintraarterially averaged every 10-second, and HR and QTc intervalderived from ECG recording averaged every 12 heart beats. The QTinterval was manually defined by one investigator and QTc was generatedfrom QT corrected by RR interval using Fridericia method(QTc=QT/RR1/3).9

Results: The body weight of control and MI rabbits at the time of studywas similar (3.35±0.19 versus 3.06±0.28 kg).

Cardiac Images Representative cardiac short-, long-axis and polar mapimages of control and MI rabbits are shown in FIG. 12. FIG. 12 showsrepresentative cardiac images of imaging agent 1 in control and chronicmyocardial infarct (MI) rabbits. These images were acquired at 20-30 minafter imaging agent 1 injection and presented in cardiac short- andlong-axis views, and polar maps. Defect areas were clearly identified inthe MI rabbit. In the control rabbit, the myocardium was clearly visiblewith uniform distribution of radioactivity and minimal backgroundinterference. In the MI rabbit, a perfusion defect area in the leftventricular wall was clearly detected in the cardiac short- andlong-axis, and polar map views.

ECG Evaluation. As shown in Table 12, baseline ECG tracing (beforeimaging agent 1 injection) recorded in lead II configuration showed anormal waveform with positive QRS complexes and T waves in the controlrabbit. In contrast, the QRS complex and T wave were negative withenlarged Q wave in the MI rabbit. The study obtained ECG tracing before,1-min and 5-min after imaging agent 1 injection in control andmyocardial infarct (MI) rabbits. Table 12 shows baseline values of QTcinterval (corrected by Fridericia method) and averaged changes from thebaseline at 1, 5, 10 and 20 min after imaging agent 1 injection incontrol and MI rabbits. Similar to control, no changes in ECG wave formand QTc interval were observed after injection in MI rabbits.

TABLE 12 Changes from baseline after injection QTc (msec) Baseline 1 min5 min 10 min 20 min Control (n = 3) 319 ± 17 2 ± 15 −1 ± 11 6 ± 20 9 ±15 MI (n = 4) 288 ± 17 8 ± 5  4 ± 6 4 ± 8  3 ± 12

However, the baseline values of QTc and HR (Table 12 and Table 13) werecomparable in these two groups. Intravenous administration of imagingagent 1 did not alter the ECG waveform, cardiac rhythm, HR and QTcinterval from the baseline values at 1-, 5-, 10- and 20-minute postinjection in either control or MI rabbits. The study, in part, showedaveraged heart rate (HR) tracings of control and myocardial infarct (MI)rabbits 5-min before and 20-min after imaging agent 1 administration.Table 13 shows baseline values of HR and averaged changes from thebaseline at 1, 5, 10 and 20 min after the injection in control and MIrabbits. Similar to control, no changes in HR were observed afterinjection in MI rabbits.

TABLE 13 Heart rate Changes from baseline after injection (beat/min)Baseline 1 min 5 min 10 min 20 min Control 159 ± 8  −4 ± 2 −2 ± 1  −2 ±4 −4 ± 4  MI 162 ± 36  6 ± 5 3 ± 8  1 ± 6 −7 ± 10

Arterial Pressure Measurement. In contrast to HR and QTc, the baselinevalues of MAP, SAP and DAP (Table 14 and Table 15) were significantlylower in MI rabbits than in control rabbits. In control rabbits,injection of imaging agent 1 did not induce changes in MAP (Table 14),SAP and DAP (Table 15). In agreement with the control animal, noalterations of these parameters were observed in the MI rabbits duringand after administration of imaging agent 1. The study, in part,demonstrated averaged mean arterial pressure (AP) tracings of controland myocardial infarct (MI) rabbits 5-min before and 20-min afterimaging agent 1 administration. Table 14 shows baseline values of meanAP and averaged changes from the baseline at 1, 5, 10 and 20 min afterthe injection in control and MI rabbits. Similar to control, no changesin mean AP were observed after injection in MI rabbits. * indicatesp<0.05 vs. control. The study, in part, demonstrated averaged systolicand diastolic arterial pressure (AP) tracings of control and myocardialinfarct (MI) rabbits 5-min before and 20-min after imaging agent 1administration. Table 15 shows baseline values of systolic and diastolicAP and averaged changes from the baseline at 1, 5, 10, and 20 min afterthe injection in control and MI rabbits. Similar to control, no changesin mean AP were observed after injection in MI rabbits. * indicatesp<0.05 vs. control.

TABLE 14 Mean AP Changes from baseline after injection (mmHg) Baseline 1min 5 min 10 min 20 min Control 89 ± 11 0 ± 0 −2 ± 0 −2 ± 3 −1 ± 6 MI 61± 6* 2 ± 1  2 ± 2 −1 ± 2  2 ± 3

TABLE 15 Changes from baseline after injection AP (mmHg) Baseline 1 min5 min 10 min 20 min Systolic AP Control 114 ± 11  0 ± 1 −2 ± 1 −1 ± 6  0± 5 M1  79 ± 11* 1 ± 2  2 ± 2 0 ± 1 1 ± 7 Diastolic AP Control 76 ± 10 0± 1 −1 ± 2 0 ± 5 0 ± 5 MI 53 ± 4* 1 ± 1  2 ± 2 1 ± 1 2 ± 2

Discussion: The study was designed to study imaging agent 1 as a PETimaging agent for evaluation of myocardial perfusion in diagnosis andprognosis of coronary heart disease. It was evaluated for safety innormal animals and imaged in animal models of acute myocardial ischemiaand MI induced by ischemia-reperfusion injury (Yu M, Guaraldi M T,Mistry M, Kagan M, McDonald J L, Drew K et al.: a novel PET myocardialperfusion imaging agent. J Nucl Cardiol 2007; 14:789-98; Nekolla S G,Reder S, Higuchi T, Dzewas G, Poethko T, Preissl A et al. Assessment ofImaging Properties of a New F-18 Labelled Flow Tracer in a Pig Model. JAm Coll Cardiol 2008; 51:A170; and Mistry M, Onthank D, Green J, CicioS, Casebier D, Robinson S et al. Toxicological Evaluation of BMS-747158,a PET Myocardial Perfusion Imaging Agent. The Toxicologist 2008;102:476). This study was designed to further assess this agent in achronic cardiac compromised animal model. The model was created bychronic ligation of coronary artery in rabbits. This rabbit model waschosen based on several characteristics: 1) Similar to humans andcompared to other species, rabbits have poor collateral circulation inthe heart and develop MI readily after sudden coronary artery occlusion(Bell D R. Special Circulations. In: Rhoades R, Bell D R, editors.Medical Physiology: Principles for Clinical Medicine. 3rd ed. 2008. p.290-304; and Maxwell M P, Hearse D J, Yellon D M. Species variation inthe coronary collateral circulation during regional myocardialischaemia: a critical determinant of the rate of evolution and extent ofmyocardial infarction. Cardiovasc Res 1987; 21:737-46). 2) Cardiacfibroblasts and regulation of collagen biosynthesis, which are criticalin wound healing after myocardial injury, in rabbits are similar to thatobserved in humans with regard to angiotensin system (Gallagher A M,Bahnson T D, Yu H, Kim N N, Printz M P. Species variability inangiotensin receptor expression by cultured cardiac fibroblasts and theinfarcted heart. Am J Physiol 1998; 274:H801-H809). 3) After coronaryligation, plasma and myocardial norepinephrine levels increase (MakinoT, Hattori Y, Matsuda N, Onozuka H, Sakuma I, Kitabatake A. Effects ofangiotensin-converting enzyme inhibition and angiotensin II type 1receptor blockade on beta-adrenoceptor signaling in heart failureproduced by myocardial Infarction in rabbits: reversal of alteredexpression of beta-adrenoceptor kinase and G i alpha. J Pharmacol ExpTher 2003; 304:370-9; and Fujii T, Yamazaki T, Akiyama T, Sano S, MoriH. Extraneuronal enzymatic degradation of myocardial interstitialnorepinephrine in the ischemic region. Cardiovasc Res 2004; 64:125-31).Norepinephrine clearance in the heart of rabbit is mainly via neuronalnorepinephrine transporter (Gao D W, Stillson C A, O'Connell J W.Absence of MIBG uptake in the denervated rabbit heart. J Nucl Med 1996;37:106p), similar to in humans (Eisenhofer G, Friberg P, Rundqvist B,Quyyumi A A, Lambert G, Kaye D M et al. Cardiac sympathetic nervefunction in congestive heart failure. Circulation 1996; 93:1667-76). 5)Species size is appropriate for high quality PET imaging in a microPETcamera while allowing concurrent ECG monitoring. In contrast to ECGwaveform in the control rabbit, a negative QRS complex with an enlargedQ wave and inverted T wave were observed in lead II configuration in theMI rabbit, indicating an abnormal ventricular depolarization andrepolarization. Following complete obstruction of a coronary arterybranch (coronary ligation), oxygen carried to the region is reduced orceased depending on collateral circulation, leading to cell death andtissue necrosis. A rapid tissue repair process is then started,including initial inflammation followed by angiogenesis, increasedfibroblast proliferation and collagen production and deposition. Thesechanges ultimately result in formation of scar tissue to 10 rebuild thenecrotic region in the heart (Abbate A, Biondi-Zoccai G G, Van Tassell BW, Baldi A. Cellular preservation therapy in acute myocardialinfarction. Am J Physiol Heart Circ Physiol 2009; 296:H563-H565; and SunY, Weber K T. Infarct scar: a dynamic tissue. Cardiovasc Res 2000;46:250-6). Histological examination has indicated that increasedfibroblast proliferation and scar formation initiate at about 2 and 18days respectively post coronary ligation in rabbits (Morales C, GonzalezG E, Rodriguez M, Bertolasi C A, Gelpi R J. Histopathologic time courseof myocardial infarct in rabbit hearts. Cardiovasc Pathol 2002;11:339-45). In present study, the formation of scar tissue in the leftventricle of our rabbits 4-week post coronary ligation is consistentwith the findings of enlarged Q wave in ECG 20 and in other similarstudies (Gonzalez G E, Palleiro J, Monroy S, Perez S, Rodriguez M,Masucci A et al. Effects of the early administration of losartan on thefunctional and morphological aspects of postmyocardial infarctionventricular remodeling in rabbits. Cardiovasc Pathol 2005; 14:88-95; andConnelly C M, Vogel W M, Wiegner A W, Osmers E L, Bing O H, Kloner R Aet al. Effects of reperfusion after coronary artery occlusion onpost-infarction scar tissue. Circ Res 1985; 57:562-77). Previously,imaging agent 1 has been demonstrated to be capable of detecting regionsof acute myocardial ischemia and necrosis induced by coronary ligationand ischemia-reperfusion injury in rats, rabbits and pigs (Yu M,Guaraldi M T, Mistry M, Kagan M, McDonald J L, Drew K et al.: a novelPET myocardial perfusion imaging agent. J Nucl Cardiol 2007; 14:789-981;Nekolla S G, Reder S, Saraste A, Higuchi T, Dzewas G, Preissel A et al.Evaluation of the novel myocardial perfusion positron-emissiontomography tracer 18F-BMS-747158-02: comparison to 13N-ammonia andvalidation with microspheres in a pig model. Circulation 2009;119:2333-42; and Higuchi T, Nekolla S G, Huisman M M, Reder S, PoethkoT, Yu M et al. A new 18F-labeled myocardial PET tracer: myocardialuptake after permanent and transient coronary occlusion in rats. J NuclMed 2008; 49:1715-22). Imaging in this study in a rabbit model ofchronic MI clearly demonstrated that imaging agent 1 imaging can detectchronic MI, possibly scar tissue suggested by ECG and other studies.imaging agent 1 has high affinity to mitochondrial complex I and, atvery high concentrations (>200 μg/kg), induces transit clinical signs,such as rapid and labored breathing, decreased activity, hunchedposture, urination, in normal rats and dogs (Mistry M, Onthank D, GreenJ, Cicio S, Casebier D, Robinson S et al. Toxicological Evaluation ofBMS-747158, a PET Myocardial Perfusion Imaging Agent. The Toxicologist2008; 102:476). However, these signs were not observed when the dose wasequal or below 100 μg/kg. In anesthetized naïve dogs, no cardiovascularchanges (MAP, HR, left ventricular contractility etc) were observedduring and after intravenously injection of imaging agent 1 at doses ofequal or less than 10 μg/kg (unpublished data). This represents a largesafety margin over the maximal clinical imaging agent 1 dose of 0.07μg/kg.

In the present study, baseline values of MAP, SAP and DAP were lower inthe MI rabbits than in control rabbits, indicating the chronic MI hadcompromised the cardiovascular system in these rabbits. The dose ofimaging agent 1 used for rabbit imaging was in the clinical formulationand approximately 0.5 mCi/kg (˜1.5 mCi in a 3-kg rabbit) which is alsoapproximately 3-fold higher than the clinical dose (total rest andstress 11 doses: ˜10 mCi in a 60 kg individual). With this dose and in acardiac compromised condition, no change in arterial pressure, heartrates and ECG waveform were produced. These findings indicate thatimaging with imaging agent 1 is safe even in a cardiac compromisedcondition.

The results show that cardiac PET imaging with imaging agent 1 detectschronic myocardial infarction (fibrosis and scar formation) in additionto myocardial ischemia and necrosis under an acute condition. At imagingdose levels, imaging agent 1 is safe to be used even in cardiaccompromised condition, at least in rabbits.

Example 31

The following example describes brain imaging of imaging agent 1 andevaluation of the blood brain barrier permeability in rats. PET imagingin animals and humans indicate this compound crosses the normal bloodbrain bather (BBB) and can image CNS disease. Studies to date have notassessed how effectively imaging agent 1 crosses the BBB. The presentstudies compared brain uptake in rats in the presence and absence of BBBdisruption.

Methods: Male Sprague-Dawley rats were anesthetized with sodiumpentobarbital and the left external carotid artery was cannulated closeto the internal and external carotid bifurcation. Using saline ascontrol and 25% D-mannitol as hypertonic solution, each were perfusedretrogradely in six animals (0.3 mL/kg/sec) for 30 seconds. Two minuteslater, ˜1 mCi imaging agent 1 was injected via tail vein and the brainwas imaged with a microPET camera for 30 minutes. Evans blue (2%, 5mL/kg) was also injected intravenously and only animals demonstratingclear BBB disruption by Evans blue staining were included in the study.Following completion of imaging, the brain was harvested, photographedand dissected into left and right hemispheres and cerebellum. The tissuecontent of imaging agent 1 radioactivity was measured by gamma counterand Evans blue levels were determined by fluorescence method forcalculation of the % injected dose/gram tissue and μg Evan blue/gramtissue, respectively.

Results: See Table 16. Infusion of 25% D-mannitol resulted in a markedincrease in Evans blue uptake in the left hemisphere (633%) and someincreased uptake in the right hemisphere (216%) and cerebellum (186%)compared to saline control. In normal rats and saline infused controlrats a high level of imaging agent 1 accumulated in the brain shortlyafter administration. PET imaging showed this high uptake of imagingagent 1 in saline control rats was only minimally increased in brainregion following BBB disruption.

Imaging agent 1 has a high BBB permeability that is only minimallyincreased following disruption and may be used for brain imaging.

TABLE 16 right hemisphere left hemisphere cerebellum Brain uptakecontrol BBBD control BBBD control BBBD Evan blue (μg/g) 19 ± 2  60 ± 8 21 ± 2  154 ± 13  29 ± 4  83 ± 12 imaging agent 1 0.72 ± 0.03 0.81 ±0.04 0.72 ± 0.03 0.93 ± 0.06 0.78 ± 0.03 0.95 ± 0.05 (% ID/g)

Example 32

The following example is related to ¹⁸F labeled imaging agent 1 PETmyocardial perfusion imaging detecting more severe and extensive stressinduced myocardial ischemia than Tc-99m Sestamibi SPECT. In this study,rest-stress Tc-99m Sestamibi SPECT and imaging agent 1 PET MPI werecompared for evaluation of stress induced myocardial perfusionabnormalities.

Methods: Thirteen patients, from a single center, underwent rest-stressTc-99m Sestamibi SPECT MPI, rest-stress imaging agent 1PET MPI andcoronary angiography. In each patient, 17 myocardial segments werevisually scored for rest and stress images by independent observers whowere blinded to all other results. For each patient, summed stressscores (SSS), summed rest scores (SRS), and summed difference scores(SDS) were determined from segmental scores. Percent narrowing in eachcoronary artery was evaluated blindly and 70% luminal diameter narrowingwas considered significant.

Results: There were 15 diseased coronary arteries; 7 left anteriordescending, 5 left circumflex and 3 right coronary arteries. Inmyocardial segments that were supplied by diseased coronary arteries,SSS and SDS were significantly higher by PET than SPECT (Table 17).

These data showed that as compared to Sestamibi SPECT, rest-stress ¹⁸Flabeled imaging agent 1 PET MPI demonstrated more severe and extensivestress induced perfusion abnormalities in myocardial regions that aresupplied by diseased coronary arteries.

TABLE 17 imaging agent 1 PET Tc-99m Sestamibi SPECT P Value SSS 16.1 ±7.8 8.6 ± 5.8 <0.001 SDS 12.3 ± 7  5.4 ± 4.2 <0.05 SRS  3.8 ± 6.6 3.1 ±3.3 NS

Example 33

The following example describes a comparison of myocardial stressperfusion defect assessment using ^(99m)Tc sestamibi SPECT versusimaging agent 1 PET. Myocardial uptake of imaging agent 1 exhibits astronger relationship with myocardial blood flow across the range ofachievable flow than ^(99m)Tc sestamibi. The assessment of myocardialperfusion defects by imaging agent 1 PET and ^(99m)Tc sestamibi SPECTwere compared.

Methods and Results: Twenty six patients (20 men) underwent SPECT andPET within 6-months. PET was performed with imaging agent 1 at rest(2.3-3.9 mCi) followed 60 min (n=18) or 24 h (n=8) later with exercise(n=16) or adenosine (n=10) stress (7.3-8.6 mCi). Image quality of SPECTand PET was consensually assessed by 2 independent blinded readers andgraded as excellent, good, or fair. Stress and rest perfusion defects onSPECT and PET were assessed by the same readers by computer-assistedvisual interpretation, using the standard 17 segment, 5 point-scoringmodel (0=normal; 4=absent uptake). The extent and severity of ischemia(summed difference score (SDS)) was derived from the difference betweensummed stress (SSS) and summed rest scores (SRS). Image quality with PETwas excellent in 24 and good in 2 patients. In contrast, there were 7excellent, 18 good, and 1 fair quality study, p<0.001 by SPECT. In 14patients with abnormal SPECT (SSS >4), mean SDS was greater with PETthan with SPECT (9.6±1.8 vs. 5.4±0.7, p=0.02). In all 12 patients withnormal SPECT (SSS <4), SDS was zero by PET and SPECT.

Compared to ^(99m)Tc sestamibi SPECT, imaging agent 1 PET providesbetter image quality and results in a significant increase in the SDS inpatients with abnormal SPECT. These results showed that PET imaging withimaging agent 1 provided better assessment of the magnitude ofmyocardial ischemia than SPECT.

Example 34

The following describes cardiac phantom simulation of dose injectionparameters for one-day rest/stress myocardial perfusion (MP1) PETimaging with imaging agent 1 tracer. A 1-day rest/stress (RS) protocolfor MPI with imaging agent 1 can create cross contamination (CC) in thestress image. A phantom simulation was conducted to assess the impact ofCC on image characteristics for a range of conditions.

Methods: A F18 phantom with myocardium (M)=0.21 uCi/ml and liver(L)=0.22, simulating normal rest, was scanned on a Siemens Biograph-64PET/CT for 30 min. It was washed and refilled with L=0.42, torso=0.09and M=0.9 with a 40% defect in septal wall, then scanned for another 30min SUV from 12 patients in a Phase II trial was used to assurerealistic simulation. Registered RS images were blended to simulate CCfor combinations of dose ratio (DR=1-5) and wait time (WT=30-120 min)between RS injections using blending coefficients determined by M-SUV,DR, rest dose decay and WT. Each blended image set was measured fordefect contrast (DC) using (SUV_(n)−SUV_(d))/SUV_(n), defect volume (DV)using pixel values ≧(SUV_(n)+SUV_(d))/2 in defect, and wall uniformity(WU) using (SD/mean) in normal wall. Degradation ≦10% for DC, DV and WUwas applied to determine the minimal WT for DR.

Results: WU (<7.6%) and DV (<2%) for any type of stress were notsignificantly affected by any combination. DC degradation was reduced tothe acceptable range by increasing DR, WT or both.

Example 35

The following describes high definition cardiac perfusion PET using anew ¹⁸F imaging agent, imaging agent 1. HD.PET technology improvesspatial resolution and signal-to-noise on reconstructed PET images (IEEETMI 2006:25:7:907-921) but the thermal path of the positron emitted byrubidium limits its benefits in ⁸²Rb perfusion images. To evaluate itsfull potential for high-resolution cardiac imaging, HD.PET withmyocardial perfusion images obtained with a new ¹⁸F based agent (imagingagent 1) was evaluated.

Methods: Images of 15 subjects in a study of imaging agent 1 perfusionagent were acquired on a 4-ring Siemens Biograph-64. Static and 8-binECG-gated images were generated using standard reconstruction (SR—2DAttenuation Weighted Ordered Subsets Expectation Maximization) andHD.PET. The wall/cavity contrast and contrast-to-noise ratio (CNR), andmaximum to defect contrast were computed. Wall thickness at threedifferent levels of heart (basal, mid, apical), wall motion, wallthickening and ejection fraction (EF) were also estimated with automaticquantification.

Results: HD.PET showed significant contrast change compared to SR(+32.3±17.9%, p<0.05). CNR also was improved with HD.PET (+26.7±22.3%vs. SR, p<0.05). The average contrast between the maximum in themyocardium and the 22 defects in the 15 patients was increased withHD.PET (4.0±1.7) compared to SR (3.2±1.2, p<0.05). The average wallthickness was 16:3±2.9 mm, 16.7±2 9 min and 15.6±2.2 min (basal, mid,apical) with SR compared with 14.7±2.8 mm, 14.1±3.0 min and 13.0±1.7 mmwith HD.PET (p<0.05). EF, wall motion and wall thickening did not showany Significant differences with HD.PET.

Conclusion: Perfusion studies with imaging agent 1 show significantlyimproved image resolution, contrast and contrast-to-noise with HD.PETreconstruction as compared with the standard reconstruction technique.

Example 36

Using tracer kinetic modeling with imaging agent 1 PET, absolutequantification of myocardial blood flow (MBF) was shown to be feasibleeven at high flow rates. The study examined whether retention and SUVcalculations were also suitable for the assessment of coronary flowreserve (CFR) in a pig model.

Methods: Nine pigs were subjected to dynamic PET imaging of 100-200 MBqimaging agent 1 at rest and stress. MBF was evaluated using both imagingagent 1 PET 3-compartmental modeling and the co-injected microspheres.Retention was calculated as uptake between 5-10 and 10-20 min divided bythe integral under the input function. Standard SUV calculation for thesame time points was also used.

Results: MBF ranged from 0.5-2.8 mL/min/g Both retention and SUV showedgood correlation with both imaging agent 1 and microsphere MBF (5-10min: r=0.69, p<0.05 and 0.69, p<0.05 for retention, r=0.86, p<0.001 and0.88, p<0.001 for SUV). Linear regression analysis revealed good resultsonly for the earlier interval (y=8.27x+1.45 and 7.11x+3.63 forretention, 1.11x+0.01 and 0.99x+0.26 for SUV), but at later interval anunderestimation was found. Calculation of stress/rest ratio forretention and SUV allows assessment of CFR. The agreement betweenretention and SUV derived CFR and both imaging agent 1 and microspheresCFR, yielded modest mean differences in the early interval (0.1 and−0.05, for retention, 0.05 and −0.09 for SUV) and larger deviations inthe late interval (˜0.47 and −0.62 for retention, −0.4 and −0.54 forSUV).

Using imaging agent 1, a simplified kinetic analysis model for theassessment of MBF index and CFR was feasible. Furthermore, SUV derivedvalues were suitable for tracer injection outside the imaging device andallowed for a physical stress test. These results provided a basis for asimplified quantitative approach in the routine clinical setting.

Example 37

The following example describes the synthesis of imaging agent precursor1, according to the scheme shown in FIG. 3.

Example 37A Synthesis of 2-(t-butyl)-4,5-dichloropyridazin-3(2H)-one(Compound 11)

Solid t-butyl hydrazine hydrochloride (1 equiv) was added to a stirredsolution of sodium hydroxide (0.95 equiv) dissolved in 10% water/toluenemixture (6 vol) at ambient temperature. The resulting white suspensionwas cooled slightly while mucochloric acid (1 equiv) was slowly added.After completion of the addition, the reaction mixture was stirred atambient temperature for 20-30 minutes followed by dropwise addition ofacetic acid (0.95 equiv). The reaction mixture was heated to 45-50° C.and stirred for 18 h, until starting material was consumed, as measuredby HPLC. The reaction solution was allowed to cool to ambienttemperature and then was diluted with water (˜7 vol) and the organiclayer separated. The organic layer was cooled to 0° C. and washed with30% NaOH (3.6 vol), followed by 35% HCl (3.6 vol) and water (2×3.6 vol).The organic solution was concentrated under vacuum and restripped withmethanol (1.5 vol) to yield compound 11 as a brown solid that was driedunder vacuum at 35° C. (65-75% yield, 100% purity by HPLC).

Example 37B Synthesis of2-(t-butyl)-4-chloro-5-((4-(hydroxymethyl)benzyl)oxy)pyridazin-3(2H)-one(Compound 13)

A solution of compound 11 (222 g) in dry dimethylformamide (780 mL) wasslowly added to a stirred mixture of 1,4-phenylenedimethanol (compound2, 690 g) and cesium carbonate (1.3 kg) in dry dimethylformamide (2.22L) heated to 65° C. The resultant mixture was stirred at 65° C. for anadditional 4 h, when the reaction was cooled and filtered. The filtratewas diluted with 5% brine and extracted with toluene. The combinedtoluene extracts were washed twice with 5% brine and the organicsconcentrated under reduced pressure. The resulting crude wascrystallized from hot methanol/water mixture, filtered, washed withmethanol/water and dried under vacuum at 40-45° C. to afford compound 3(224 g) as an off-white powder in 69% yield, contaminated with 6% of theproduct of dialkylation of compound 12 with compound 11.

Example 37C Synthesis of5-((4-(bromomethyl)benzyl)oxy)-2-(t-butyl)-4-chloropyridazin-3(2H)-one(Compound 14)

A dry vessel was charged with anhydrous dichloromethane (670 mL) andcompound 13 (224 g). A 1.0M solution of phosphorous tribromide indichloromethane (345 mL) was added to the mixture over 30 min at 25° C.and the solution stirred for another 30 min. The reaction was dilutedwith dichloromethane (450 mL) and water (670 mL), the layers separated,and the aqueous phase extracted with dichloromethane (670 mL). Thecombined organic layers were washed twice with 5% brine, concentratedunder vacuum, and dried for 34 h under vacuum at 40° C. to yieldcompound 14 as an off-white solid (258 g, 96% yield).

Example 37D Synthesis of2-(t-butyl)-4-chloro-5-((4-((2-hydroxyethoxy)methyl)benzyl)oxy)pyridazin-3(2H)-one (Compound 15)

Ethylene glycol (2.9 L) was charged into a dry vessel and treated withsolid potassium t-but oxide (74 g). The suspension was heated to 60° C.to form a solution and then cooled to 20-25° C. A solution of compound14 (290 g) in dry THF (1.45 L) was added in one portion to the stirringethylene glycoside solution. The resultant mixture was heated to 60° C.and stirred at this temperature for 16.5 h when it was then cooled to25° C. and diluted with water (2.9 L) and toluene (4.35 L). The organiclayer was separated, washed three times with water and concentratedunder vacuum. Another charge of toluene (4.35 L) was added andconcentrated under vacuum again to afford crude compound 15 as a brownviscous oil (260 g, 95% yield) Crude compound 15 (690 g) was dissolvedin dichloromethane (0.5 kg/L) and purified by chromatography (silicacolumn, 1:1 heptanes/ethyl acetate, flow rate=6 L/min, 10 L fractions).The combined fractions were combined and concentrated under vacuum toafford compound 15 as a clear, viscous oil (520 g, 70% yield).

Example 37D-1

The following example describes the synthesis of compound 15, using analternate synthetic method relative to Example 37D. Into a clean, dryreactor equipped with overhead stirrer and temperature probe was chargedanhydrous ethylene glycol (2900 mL), followed by potassium t-but oxide(42.2 g) at ambient temperature. The solution was heated to 55 to 60° C.to form a clear solution of the ethylene glycoside and then cooled to20° C. to 30° C. under an inert atmosphere. This solution was assayedfor total base content. A separate vessel was charged with anhydroustetrahydrofuran (725 mL) and compound 14 (145 g) with stiffing to form asolution at ambient temperature. This solution was added in a singleportion directly to the ethylene glycoside solution at 20 to 30° C. Themixture was heated to 60° C. and stirred at this temperature. When thereaction was complete, it was cooled to 20° C. and toluene (2200 mL) andwater (2200 mL) were added with stirring to form two layers when allowedto settle. The layers were separated and the organic layer washed with2200 mL each of sodium bicarbonate solution and water (twice). Theorganic layer was concentrated at <50° C. under vacuum to give compound15 as a viscous oil (133.4 g, 91% when corrected for residual toluene).

Example 37E Synthesis of Contrast Agent Precursor 1

A dry reactor was charged sequentially with dichloromethane (6.6 L),compound 15 (510 g) dissolved in dichloromethane (1.1 L), triethylamine(0.25 L), p-toluenesulfonyl chloride (305 g), and dimethylaminopyridine(7 g). The solution was stirred at ambient temperature for 28 h when itwas washed with 1.0 M HCl (2×10 L), water (10 L), 5% sodium bicarbonate(2×10 L), and water (10 L). The organic solution was filtered anddichloromethane removed under reduced pressure to afford imaging agentprecursor 1 as a thick oil.

Crude imaging agent precursor 1 (21.5 g) was added to cumene (125 mL)and heated to 60° C. to dissolve the solids. It was cooled to 40° C. and1% w/w imaging agent precursor 1 crystals added to seed thecrystallization. The solution was held for 3 h at 35° C. to allowcrystallization, and then cooled to ambient temperature and stiffed for6 h to complete crystallization. The solids were filtered, dried brieflyunder vacuum, and then added to isobutyl acetate (125 mL). After heatingto 70° C., the solids dissolved, and the solution was then cooled to40-50° C. and seeded with 1% w/w imaging agent precursor 1. Afterholding at 40-50° C. for five hours, the slurry was cooled to ambienttemperature over 2 hours and held for 12 h. The resulting solids werefiltered, rinsed with cold isobutyl acetate, and dried under vacuum toafford 12.8 g of imaging agent precursor 1 (60% from compound 15).

In some cases, the triethylamine stoichiometry was increased from about1.15 to about 1.40 equiv. In some cases, the p-toluenesulfonyl chloridestoichiometry was increased from about 1.15 to about 1.20 equiv. In somecases, the dimethylaminopyridine stoichiometry was increased from about0.04 to about 0.10 equiv.

In some embodiments, the cumene crystallization was completed under thefollowing conditions: Dilution: 10.0 volumes; Seeding temperature: 45°C.; Crystallization hold time at seeding temperature: 3 h; Cooling rate:5° C./h; Granulation temperature: 20° C.; Granulation time: >3 h;Filtration temperature: 20° C.

In other embodiments, the cumene crystallization was completed under thefollowing conditions: Dilution: 6.5 volumes; Seeding temperature: 50°C.; Crystallization hold time at seeding temperature: 6 h; Cooling rate:10° C./h; Granulation temperature: 10° C.; Granulation time: >8 h;Filtration temperature: 10° C.

In a certain embodiment, compound 16 (20.0 g) was suspended in cumene(6.5 volumes) then warmed to 68° C. The resulting solution was cooled to50° C. then seeded with compound 16; slow formation of a precipitate wasobserved. The resulting suspension was held at 50° C. for 6 h thencooled at 10° C./h to 10° C., maintained 12 h, filtered and washed.Following in vacuo drying at 60° C., 16.4 g of compound 6 was obtained(82% recovery; 96% solvent and purity adjusted).

In some embodiments, the isobutyl actetate crystallization was conductedunder the following conditions: Dilution: 8 volumes; Seedingtemperature: 50° C.; Crystallization hold time at seeding temperature: 3h; Cooling rate: 5° C. per hour; Granulation temperature: 20° C.;Granulation time: >10 h; Filtration temperature: 20° C.

In other embodiments, the isobutyl actetate crystallization wasconducted under the following conditions: Dilution: 5 volumes; Seedingtemperature: 48° C.; Crystallization hold time at seeding temperature:10 h; Cooling rate: 2.5° C./h; Granulation time: 0 h; Filtrationtemperature: 10° C.

In a certain embodiment, cumene crystallized compound 16 (15.40 g) wassuspended in isobutyl acetate (5 volumes) then warmed to 68° C. Theresulting solution was cooled to 48° C. then seeded with BMS-747155-01(0.1% w/w); immediate formation of a precipitate was observed. Theresulting suspension was held at 48° C. for 10 h then cooled at 2.5°C./h to 10° C., filtered and washed. Following in vacuo drying at 60°C., 13.10 g of compound 16 was obtained (85% recovery) which passed allspecifications.

Example 38

The following example describes an alternate route to synthesizing2-(t-butyl)-4-chloro-5-((4-(hydroxymethyl)benzyl)oxy)pyridazin-3(2H)-one (compound 13), as shown in FIG. 4.

Example 38A Synthesis of2-(t-butyl)-4-chloro-5-hydroxypyridazin-3(2H)-one (Compound 17)

A dry vessel was sequentially charged while stirring with compound 11(100 g), potassium hydroxide (76.1 g), and ethylene glycol (1 L). Theresulting suspension was heated to 115° C. and stirred at thistemperature for 5 hrs. The brown solution was cooled to 0° C. and 1 Mhydrochloric acid solution (1 L) added slowly with stirring over 60minutes, keeping the temperature below 25° C. during the addition,resulting in precipitation of a light brown solid. The slurry wasstirred for 2 hrs and filtered, washing the cake with cold water (4×500mL) and ethanol (100 mL). The Crude compound 17 thus obtained was thenrecrystallized from hot ethanol (1 L), filtered, and dried under vacuumfor 34 h at 45° C. to yield pure compound 17 (68.3 g, 75% yield).

Example 38B Synthesis of methyl4-(((1-(t-butyl)-5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)oxy)methyl)benzoate(Compound 18)

A dry vessel under nitrogen atmosphere was charged sequentially withcompound 17 (66 g), dimethylformamide (660 mL) and potassium carbonate(45 g). To this was added methyl 4-(bromomethyl)benzoate (78 g) and theresulting suspension stirred for 18 h at 20° C. Water (700 mL) was addedover 30 minutes to precipitate the product and dissolve remaining salts.The slurry was stirred for 1.5 h and the resulting solids filtered,washed with water (4×300 mL) and cyclohexane (2×150 mL) and dried undervacuum at 45° C. to afford compound 18 (112.8 g, 99%) as a white powder.

Example 38C Alternate synthesis of2-(t-butyl)-4-chloro-5-((4-(hydroxymethyl)benzyl)oxy)pyridazin-3(2H)-one (Compound 13)

A dry vessel with overhead agitation under an atmosphere of dry nitrogenwas charged sequentially with 2-methyltetrahydrofuran (500 mL) andcompound 18 (50 g) at ambient temperature. The resulting suspension wascooled to −7° C. and a solution of diisobutylaluminum hydride in toluene(1.5M, 119 mL) added drop-wise over 1 h keeping the temperature below 3°C. After stirring for 1.5 h at −5° C.-0° C., the reaction was quenchedby addition of propan-2-ol (50 mL) at a rate to keep the temperaturebelow 4° C. The quenched reaction mixture was then added dropwise to asolution of hydrochloric acid (2M, 500 mL) over 75 min, keeping thetemperature below 7° C. The biphasic solution was warmed to 22° C. andthe layers separated. The organic layer was then washed with 500 mL eachof 2M hydrochloric acid, saturated sodium bicarbonate solution and waterand then concentrated under reduced pressure to afford crude compound 13as an off-white solid (42.4 g). This was recrystallized from hotisopropyl acetate (200 mL), seeding the solution at 65° C. and holdingat this temperature for one h, followed by cooling to 0° C. over 4 h.The resulting white solid was filtered and dried under vacuum at 45° C.to afford compound 13 (35 g, 76% yield).

In some cases, the above experiment was performed with both lithiumaluminum hydride and sodium bis(2-methoxyethyoxy)aluminum hydride (RedAl) as well as diisobutylaluminum hydride (DIBAL-H). In some cases,solutions of DIBAL-H in dichloromethane, toluene, and hexane wereemployed. In some cases, selection of 2-MeTHF (vs. THF) as co-solventwas performed due to its reduced aqueous solubility. In some cases,stress studies revealed the DIBAL reduction performed well, inparticular, at temperatures between ˜15 to +10° C. In some cases, theDIBAL-H was charged in two portions; 2.20 equiv followed by additionalreagent if incomplete reaction was observed. In some cases, residualwater was found to have hydrolyzed DIBAL-H and the impurity profileremained unchanged.

In some embodiments, the reaction was conducted under the followingconditions: −15 to +10° C.; up to ˜2.35 equiv DIBAL-H; up to 5% H₂O(w/w) in precursor; <0.75% precursor remaining at full conversion.

Example 39

The following example described an alternate synthetic route to2-(t-butyl)-4-chloro-5-((4-((2-hydroxyethoxy)methyl)benzyl)oxy)pyridazin-3(2H)-one (Compound 15), as shown in FIG. 5.

Example 39A Preparation of methyl 4-(1,3-dioxolan-2-yl)benzoate(compound 19)

Methyl 4-formylbenzoate (3.28 g, 20.0 mmol) was suspended in ethyleneglycol (4.46 mL, 80.0 mmol), then successively treated with triethylorthoformate (3.66 mL, 22.0 mmol) and Me₃NPhBr₃ (376 mg, 1.00 mmol) at22° C.; within 5 min, all solids dissolved. The resulting orangesolution was stirred 0.5 h, then diluted with saturated aqueous NaHCO₃(50 mL), transferred to separatory funnel and washed with EtOAc (3×50mL). The combined EtOAc washes were dried over MgSO₄, filtered andconcentrated in vacuo to a colorless oil (R_(f) 0.4 in 4:1pentane/EtOAc, KMnO₄). This material was used without furtherpurification in the subsequent reduction step.

Example 39B Preparation of (4-(1,3-dioxolan-2-yl)phenyl)methanol(Compound 20)

The crude acetal (20.0 mmol theoretical) was dissolved in dry THF (100.0mL), cooled to 0° C. and treated with LiAlH₄ (20.00 mmol; 20.00 mL of a1.0 M solution in THF) at a rate of 1.0 mL/min using a syringe pump.Upon completion of the addition, excess LiAlH₄ was consumed by thecareful addition of H₂O (800 μL). CAUTION: vigorous gas evolution! Theresulting white suspension was successively treated with 15% aqueousNaOH (800 μL) and H₂O (2.40 mL), then stirred 0.5 h to a fine whiteslurry. The solids were removed by filtration through a pad of Celitethen exhaustively washed with Et₂O. The combined filtrates wereconcentrated in vacuo to a colorless oil and purified by chromatographyon silica (50×175 mm) using 1:1 pentane/EtOAc. The main product peakeluting 470-790 mL was collected, pooled and concentrated in vacuo to acolorless oil, which solidified in the freezer (2.46 g, 13.7 mmol; 68.3%over two steps).

Example 39C Synthesis of (4-(1,3-dioxolan-2-yl)phenyl)methanol (Compound20)

Methyl 4-formylbenzoate (4.92 g, 30.0 mmol) was dissolved in dry toluene(50.0 mL), successively treated with ethylene glycol (1.84 mL, 33.0mmol) and p-TsOH.H₂O (57.1 mg, 0.30 mmol), then heated to reflux underDean-Stark conditions; acetal formation was complete within 1 h. Thesolution was then cooled to 22° C. and treated with sodiumbis(2-methoxyethoxy)aluminum hydride (45.0 mmol; 12.7 mL of a 70.3 wt. %solution in toluene) at a rate of 0.5 mL/min using a syringe pump.CAUTION: vigorous gas evolution! Upon completion of the addition, theresulting solution was further cooled to 0° C., carefully treated with asaturated aqueous solution of K,Na-tartrate (100 ml), then vigorouslystirred 1 h; steady formation of a clear solution was observed. Theresulting biphase was then diluted with EtOAc (50 mL), with transfer toa conical funnel, and the layers separated. The aqueous layer was thenwashed with EtOAc (3×50 mL) and the combined EtOAc and toluene solutionsdried over MgSO₄, filtered and concentrated in vacuo to a colorless oil.The crude product was then purified by chromatography on silica (50×135mm) using 1:1 pentane/EtOAc. The main product peak eluting 425-725 mLwas collected, pooled and concentrated in vacuo to a colorless oil,which solidified in the freezer (4.50 g, 83.2% over two steps).

Example 39D Synthesis of2-(t-butyl)-4-chloro-5-[(4-(1,3-dioxolan-2-yl)phenyl)methoxy]-2-hydropyridazin-3-one(Compound 21)

A solution of 2-(t-butyl)-4,5-dichloro-2-hydropyridazin-3-one (829 mg,3.75 mmol) and the compound 10 (451 mg, 2.50 mmol) in dry DMF (12.5 mL)was treated with Cs₂CO₃ (1.63 g, 5.00 mmol) in one portion at 22° C. Theresulting suspension was then immersed in a pre-heated oil bath (65° C.)and maintained 6 h with vigorous stirring. After cooling to ambienttemperature, the suspension was partitioned between EtOAc and H₂O (50 mLeach), with transfer to a conical funnel, and the layers separated. Theremaining aqueous layer was washed with additional EtOAc (3×50 mL) thendiscarded. The combined EtOAc solutions were further washed withsaturated aqueous NaCl (5×50 mL), then dried over MgSO₄, filtered andconcentrated in vacuo to an off-white solid. In some cases, triturationwith several small volumes of pentane was performed to generate thesolid. The crude product was then recrystallized from hot EtOAc/hexanesto afford colorless needles that were collected on a scintered glassfunnel of medium porosity, exhaustively washed with pentane and dried invacuo (573 mg, 62.8%).

Example 39E Synthesis of2-(t-butyl)-4-chloro-5-[(4-(1,3-dioxolan-2-yl)phenyl)methoxy]-2-hydropyridazin-3-one(Compound 21)

To a vessel charged with (4-(1,3-dioxolan-2-yl)phenyl)methanol (20 g,110 mmol), benzyltriethylammonium chloride (2.27 g, 10 mmol), toluene(100 mL) and sodium hydroxide (50% in water, 22 mL, 420 mmol) was addeda solution of 2-(t-butyl)-4,5-dichloro-2-hydropyridazin-3-one (22.1 g,100 mmol) in toluene (100 mL) over 5 min A gradual and acceleratingexotherm occurred with the final internal temperature reaching 39° C.After 2.5 h stiffing was halted and MTBE (50 mL) and water (100 mL)added. The phases were split and the organic layer was washed with water(100 mL) and brine (100 mL). The organic extracts were dried (MgSO₄),filtered, and concentrated under vacuum to afford a tan solid (39 g).The solids were slurried in toluene/heptanes (430 mL, 1:1) at 40° C. for2 h, cooled to ambient temperature, filtered and dried under vacuum at40° C. for 24 h (29.7 g, 69%).

Example 39F Synthesis of2-(t-butyl)-4-chloro-5-({4-[(2-hydroxyethoxy)methyl]phenyl}methoxy)-2-hydropyridazin-3-one(Compound 15)

A solution of compound 21 (365 mg, 1.00 mmol) in dry CH₂Cl₂ (10.0 mL)was cooled to −40° C. using a dry ice/MeCN bath, then treated withDIBAL-H (4.00 mmol; 4.00 mL of a 1.0 M solution in CH₂Cl₂) at a rate of0.25 mL/min using a syringe pump. The solution was maintained 1 h withperiodic addition of dry ice to the cooling bath, then carefully treatedwith wet MeOH (1 mL) and warmed to 22° C. The resulting solution wasdiluted with EtOAc (20 mL), treated with an equal volume of saturatedaqueous K,Na-tartrate, then vigorously stirred 1 h; steady formation ofa clear solution should be observed. The resulting biphase was furtherdiluted with H₂O (50 mL), with transfer to a conical funnel, and thelayers separated. The aqueous layer was then washed with EtOAc (3×50 mL)and discarded. The combined EtOAc washes were dried over MgSO₄, filteredand concentrated in vacuo to a colorless oil (R_(f) 0.2 in 1:1pentane/EtOAc, KMnO₄). The crude product was purified by chromatographyon silica (30×190 mm) using a step gradient from 1:1 pentane/EtOAc (250mL) to 3:2 pentane/EtOAc (500 mL). The main product eluting between415-580 mL was collected, pooled and concentrated in vacuo to acolorless oil (286 mg, 0.780 mmol; 78.0%).

Example 40 Synthesis of2-((4-(((1-(t-butyl)-5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)oxy)methyl)benzyl)oxy)ethyl4-methylbenzenesulfonate (imaging agent precursor 1)

A dry reactor was charged sequentially with dichloromethane (6.6 L),compound 15 (510 g) dissolved in dichloromethane (1.1 L), triethylamine(0.25 L), p-toluenesulfonyl chloride (305 g), and dimethylaminopyridine(7 g). The solution was stirred at ambient temperature for 28 h when itwas washed with 1.0M HCl (2×10 L), water (10 L), 5% sodium bicarbonate(2×10 L), and water (10 L). The organic solution was filtered anddichloromethane exchanged for ethyl acetate. The product wascrystallized from hot 1:1 heptanes/ethyl acetate (˜11 L). by slowlycooling to 0-5° C. The resulting solids were filtered, washed with coldethyl acetate/heptanes and dried under vacuum at 40° C. for 42 h toyield imaging agent precursor 1 (555 g, 77% yield).

Example 41

The following describes remote camera qualification (RCQ) of PET andPET/CT scanners for imaging agent 1 myocardial perfusion using astandardized phantom procedure.

As will be known to those of ordinary skill in the art, in a medicalimaging clinical trial, camera qualification is a critical step inassessing whether individual clinical site (CS) possesses the capabilityto execute the protocol. In some cases, a challenge lies in how tostandardize a task-specific phantom and the associated qualificationprocedure that can effectively determine if specific site scanners meetthe study requirement to join the trial.

Methods. Using various cameras, the RCQ procedure with an imaging manualcustomized for each scanner model utilized step-by-step instruction forCS to follow. A low-cost, standardized phantom using a 2 liter sodabottle with an acrylic rod (L=21 cm, D=2 cm) sealed inside the cap wasprovided to each CS (see Example 42 for more details). CS injected 3-4mCi F18 solution to the water-filled phantom to acquire image data andto test existing cardiac misregistration (MR) correction software ineach system. The RCQ procedure was performed by the CS with telephonesupport as necessary. All image data was sent to the imaging corelaboratory to analyze in terms of quantitative imaging parameters.Minimum performance criteria were established to identify cameras whoseperformance was inconsistent with accepted norms. The results are shownin Table 18.

TABLE 18 Noise Spatial Accuracy of Consistency of Consistency ofVariation of Image Resolution Image Calibration Dynamic Acq. Gated Acq.Gated Acq. Contrast (mm) Uniformity Factor Dedicated PET 93.36% ± 2.72%98.25% ± 1.88% 19.94% ± 6.18% 0.91 ± 0.04 8.36 ± 1.40 2.58% ± 0.86% 64.62% ± 20.00% (no MR) PET/CT 93.57% ± 3.96% 98.24% ± 0.66% 14.30% ±5.41% 0.97 ± 0.03 6.77 ± 1.20 2.58% ± 0.46% 93.66% ± 3.84% (no MR)PET/CT 93.70% ± 3.60% 98.05% ± 0.91% 16.02% ± 4.44% 0.95 ± 0.03 7.42 ±0.75 3.18% ± 0.56% 94.37% ± 2.87% (MR correction) Minimal >85% >85%<25% >0.9 <10 mm <5% >90% requirement

Conclusions. Remote camera qualification when integrated with astandardized phantom, comprehensive imaging manuals, full technicalsupport and centralized data analysis can be a cost-effective andefficient method to assess the performance of PET and PET/CT scanners ina large clinical trial.

Example 42

The following example describes a low cost refillable phantom forstandardization of PET Scanners

Standardization and harmonization of imaging methodology and scannerperformance is critical to the success of clinical studies using PET(e.g., as described in Example 41). Generally, this may be accomplishedwith a test object called a phantom that is loaded with an appropriatequantity of radioactive material and imaged in the same way with eachscanner. Phantoms may be constructed either of solid materials withlong-lived positron-emitters embedded or they may be filled with waterand short-lived radioactivity added as needed. Differences in observedimaging performance allow adjustment of methods or repair of equipmentas necessary to assure uniformity of image quality among all systemsused. Conventional phantoms, both solid and refillable are sufficientlyexpensive that the cost of simultaneous assessment at a large number ofsites is prohibitive. The device described in this example is a simpletask-specific phantom for cardiac PET using readily available materialsthat can be constructed for approximately 1% the price of a conventionalrefillable phantom. When combined with routine quality control, itallows the simultaneous characterization of a large number of PET andPET-CT systems for standardization in PET cardiac clinical trials.

Materials and Methods. The phantom was constructed from a standard2-liter soda bottle. A rod of acrylic plastic 8¼inches long and ¾inchesin diameter was centered and fixed to the inside of the cap of thebottle using an external screw. The surface between the end of the rodand the inside of the cap and under the screw head was sealed with glueappropriate to the materials prior to final tightening of the screw andthe phantom tested for leakage.

The phantom was filled in the following way:

-   -   1. The phantom was placed on an absorbent surface or,        preferably, in a sink and filled the phantom with tap water to        the top. Bubbles were minimized using a slow rate of water flow        into the bottle.    -   2. The acrylic rod (attached to the cap) was inserted into the        soda bottle fully and the cap screwed in place. The entire        overflow was allowed to be removed from the phantom. It was        important not to squeeze the phantom while doing this. The cap        was unscrewed and the rod slowly removed to allow any water        clinging to it to drain back into the phantom.    -   3. A clean syringe was used to draw 2 ml of water from the        phantom. Approximately 10 drops of liquid soap was added into        the phantom to prevent FDG or other F18 compounds from sticking        to the inner surface of bottle or the rod. The phantom was        shaken by tilting it up and down vertically for at least 30 sec        to ensure uniform distribution of liquid soap.    -   4. ¹⁸F activity (3-4 mCi) and the volume (several ml) in a        syringe was measured and then recorded, including volume and        assay time.    -   5. ¹⁸F was injected into the phantom slowly and the syringe        drawn back and forth to flush the remaining activity from the        syringe vigorously three times.    -   6. The same syringe was used to carefully draw out a volume of        liquid from the phantom equal to the volume plus 1 ml of the ¹⁸F        solution injected into it. This ensured the solution will not        overflow when the rod is replaced and will include a small        bubble to facilitate mixing.    -   7. The ¹⁸F activity in the syringe was measured and the        radioactivity and assay time was recorded.    -   8. The rod was reinserted into the phantom and the cap was        hand-tightened in place and ensured that it was secure and        leak-free.    -   9. The surface of the phantom was wiped with a paper towel which        was checked for radioactive contamination prior to discarding.    -   10. Image data was then acquired using any PET scanner that was        to be evaluated, under any conditions or acquisition settings        that are to be evaluated.        -   The resulting image data were assessed using conventional            tools. A large region of interest covering the central 60%            of several slices that do not include the acrylic rod may be            used to determine the degree of uniformity and correctness            of calibration factors. Region-of-interest analysis with one            or more slices containing the acrylic rod may also be used            to determine the contrast between the radioactivity-filled            volume and the area within the acrylic rod from which            radioactivity is excluded. Integration of a line profile            including the edge between the rod and the liquid may be            used to assess resolution. A variety of other factors may            also be assessed, including calibration linearity and the            capacity and accuracy of PET-CT mismatch correction using            appropriate data acquisition.

Example 43

The following example describes a comparison of imaging agent 1 and 18Ffluorodeoxyglucose (FDG) for assessment of left ventricular viabilityfollowing myocardial infarction in rats.

¹⁸F fluorodeoxyglucose (FDG) imaging of the heart is used to assessmyocardial viability. This example describes a comparison of the volumeof viable tissue in the left ventricle of normal and myocardialinfarcted (MI) rats determined by imaging agent 1 imaging with thatdetected by FDG imaging.

Methods. MI was induced in rats by 30 minutes of\ coronary occlusionfollowed reperfusion. Imaging agent 1 (1 mCi) and FDG (1 mCi) cardiacimaging in 2 days apart was performed in rats of before, two days (earlyMI) and four weeks (late MI) post surgery. A regimen of glucose andinsulin was injected before FDG imaging to ensure high cardiac uptake.Viable left ventricle was quantified in images as the volume with >50%of maximum activity.

Results. In control rats, cardiac imaging with both imaging agent 1 andFDG showed well-defined left ventricular wall and the left ventricularvolume was measured as 1.17±0.04 and 1.11±0.07 cm3 respectively beforesurgery. In early and late stage MI rats, the myocardial defect area wasclearly identified by imaging with both agents. The viable leftventricular tissue volume measured with imaging agent 1 was slightlylarger than the viable tissue area measured with FDG (0.94±0.01 vs.0.75±0.04 and 1.18±0.04 vs. 0.99±0.09 cm3 at early and late stage MI).In addition, imaging agent 1 imaging showed similar detectable leftventricular areas at 20 and 80 minutes post injection (no refill-in) inboth early and late stage MI. This example shows that imaging agent 1has the potential to be used to assess myocardial viability, like FDG,however without the need for insulin pre-treatment.

Example 44

The following demonstrates that a quantitative and perceived defectseverity are proportional with imaging agent 1 PET myocardial perfusionimaging.

In order to identify the minimum rest dose of imaging agent 1, acomparison was made between the count-related variation in normalmyocardium and the minimum change in defect severity that would resultin 50% probability of a reader's changing a segmental score by 1. Inorder to determine this limiting change in defect severity, a comparisonwas made between reader scores from a blinded read and the correspondingquantitative defect severity.

Method. Patients selected for one or more at least partially reversibledefects on SPECT studies were evaluated as part the first cohort in thePhase 2 study of imaging agent 1. Rest and stress images were read by apanel of three blinded readers. Reader′ scores using a 17-segment modelfrom the rest image data only derived from the first 20 patients werecompared with the percentage decreases from the maximum value in eachimage as calculated by standard cardiac MPI analysis software (CedarsQPS). The values were plotted and a linear regression calculated for thedata from each reader (see FIG. 13).

Results. Although there was significant range in quantitative severityvalues at each reader score value (% SD ˜20% of image maximum), the datawas well modeled with the simple linear regression resulting in R²values of 1.00, 0.978 and 0.984 for readers 1, 2 and 3 respectively. Theintercept values were 84.18%, 82.33% and 84.96% respectively while theslopes were −13.8, −9.86 and −8.53 respectively.

Discussion. These results suggest that, at least with imaging agent 1,it may be possible to estimate reader responses using a simple linearrelationship and the quantitative fraction of the maximum value withoutthe need for a normal database. Based on a mean slope of −10.7, it wasestimated that a 50% probability of a change of 1 in reader scorecorresponded to a change in quantitative severity by 5.4%

Example 45

The following examples describes a comparison of imaging agent 1 andTc-99m labeled SPECT myocardial perfusion imaging for identifyingseverity and extent of stress induced myocardial ischemia in Phase 2clinical trials

In this multi-center Phase 2 study, imaging agent 1 and Tc-99m labeledSPECT rest-stress MPI were compared for evaluation of stress inducedmyocardial perfusion abnormalities in patients (Pts) with coronaryartery disease (CAD).

Methods: 84 Pts from 21 centers, presenting with an intermediate to highCAD pre-test likelihood, underwent rest-stress Tc-99m labeled SPECT MPI,imaging agent 1 PET MPI and coronary angiography. Their mean age was64.5 years (range: 36-85) and 68 were males. In each patient, 17myocardial segments were visually scored on rest and stress images by 3independent, blinded readers. For each pt, summed difference scores(SDS) were determined from segmental scores. Percent narrowing in eachcoronary artery was quantitatively and blindly determined and >50%luminal diameter narrowing was considered significant. Of the 84 Pts, 52had CAD and 32 had insignificant CAD/normal coronary arteries.

Results. There were 105 diseased coronary arteries in 52 patients; 40left anterior descending, 30 left circumflex and 35 right coronaryarteries. In patients with at least one diseased artery, the mean (SD)PET SDS score ranged among the three readers from 6.8 (5.75) to 9.4(7.51) and the mean (SD) SPECT SDS score ranged from 4.1 (4.75) to 5.7(6.51). The differences in SDS scores between PET and SPECT werestatistically significant in all readers (p<0.01). In 52 patients withmultivessel disease and multi-readers, the adjusted mean PET SDS scorewas significantly higher than that of SPECT SDS score (p<0.001).

Conclusions. These data suggest that as compared to Tc-99m SPECT,rest-stress imaging agent 1 PET MPI demonstrates more severe andextensive stress induced perfusion abnormalities in myocardial regionsthat are supplied by diseased coronary arteries.

Example 46

The following example describes a Phase 2 clinical comparison of imagingagent 1 injection PET and Tc-99m labeled SPECT myocardial perfusionimaging for diagnosis of coronary artery disease.

In the Phase 2 study, the clinical safety of imaging agent 1 injectionwas evaluated and its diagnostic performance for detection of coronaryartery disease (CAD) was compared to rest-stress Tc-99m labeled SPECTMPI.

Methods. 143 patients (Pts) from 21 centers, presenting with a broadspectrum of CAD pre-test likelihoods, underwent rest-stress Tc-99mlabeled SPECT MPI and imaging agent 1 PET MPI. 84/143 who had anintermediate to high CAD likelihood underwent coronary angiography.Their mean age was 64.5 (range: 36-85) years and 68 were males. %narrowing in each coronary artery was quantified blindly. 52/84 Pts hadsignificant CAD (>50% luminal diameter narrowing) and 32/84 hadinsignificant CAD/normal coronary arteries. In each patient, 17myocardial segments were visually scored on rest and stress images by 3independent, blinded readers and majority rule interpretation wasdetermined in each patient for both PET and SPECT studies. Diagnosticperformance of PET was compared to that of SPECT using ROC analysis.

Results. A significantly higher % of images were rated as eitherexcellent or good on PET vs SPECT stress images (99.2% vs. 88.8%,p<0.01) and rest images (96.8% vs. 64.8%, p<0.01). Diagnostic certaintyof interpretation (% of cases with definitely abnormal/normalinterpretation) was significantly higher for PET vs. SPECT (92.0% vs.76.8%, P<0.01). The area under the ROC curve for overall diagnosis ofCAD was significantly higher for PET vs. SPECT (0.79±0.05 vs. 0.67±0.05,p<0.05). 61/143 patients reported 100 treatment emergent adverse events(AEs). Of these, 7 AEs reported in 2 patients were judged to be relatedto the study drag, but none were serious. No clinical laboratory changesfrom baseline were reported as TEAEs or considered as clinicallysignificant. The ECG data at rest revealed no evidence of any clinicallyrelevant effect on heart rate, atrio-ventricular conduction (PRinterval), depolarization (QRS duration) or repolarization (QTcFduration).

Conclusions. Within this Phase 2 clinical trial imaging agent 1 appearedto be safe and superior to Tc-99m labeled SPECT with respect to imagequality, certainty of image interpretation and overall diagnosis of CAD.

Example 47

The following described a streamlined quantification of absolutemyocardial blood flow at rest and stress with imaging agent 1 injectionPET in normal subjects and patients with coronary artery disease.

Objectives. The feasibility of streamlined quantification of rest (R)and stress (S) myocardial blood flows (MBFs) and coronary flow reserve(CFR) with imaging agent 1 for clinical use in normal subjects andcoronary artery disease (CAD) patients (Pts) was evaluated.

Methods. Ten Pts [6 with a low likelihood of CAD and 4 with CAD (>50%stenosis) and reversible defects] received imaging agent 1 injection atRand at peak adenosine S followed by 10-min dynamic acquisition. The R-Simaging protocol was same-day in 5 Pts and separate-day in 5 Pts. Rand Spolar maps were automatically generated from summed dynamic scans (0.5-2min post injection) and the 3 coronary territories (LAD, RCA, LCX) andthe left ventricular blood pool (LV) were defined automatically.Reversible defects were manually assigned on the polar maps, from whichtime activity curves (TACs) were generated. A single-compartment modelthat included an irreversible uptake constant (K) and a spillover fromblood pool activity was used to fit the tissue TACs at early times (0-2min). LV TAC was used as the input function. Recovery coefficient due topartial volume effect of myocardium was estimated as (1-spf), with spfdenoting the blood spillover fraction determined from model fitting. Thefirst pass extraction fraction for imaging agent 1 in humans was assumedto be 0.94 equivalent to that observed in pre-clinical studies. CFR wascalculated as S/R MBF.

Results. MBF and CFR were compared between 18 normal territories (in 6low likelihood Pts) and 5 reversible defect territories which weresupplied by CAD coronaries (Table 19, *=p<0.05). The results are inagreement with the published literature using N-13 ammonia PET.

TABLE 19 Normal CAD LAD RCA LCX Reversible defect RMBF 0.76 ± 0.15 0.75± 0.17 0.74 ± 0.10 0.69 ± 0.22  SMBF 2.48 ± 0.50 2.78 ± 0.43 2.66 ± 0.621/12 ± 0.19* CFR 3.25 ± 0.25 3.72 ± 0.49 3.60 ± 0.86 1.71 ± 0.41*

Conclusions. Quantification of MBF using imaging agent 1 injection PETmyocardial perfusion imaging can be streamlined in clinical applicationsto give robust MBF results.

Example 48 Synthesis of5-((4-((2-bromoethoxy)methyl)benzyl)oxy)-2-(t-butyl)-4-chloropyridazin-3(2H)-one

A solution of imaging agent precursor 1 (0.521 g, 1.00 mmol) in dryacetone (10.0 mL) was treated with LiBr (0.261 g, 3.00 mmol) in oneportion at 22° C. then warmed to 56° C. and maintained 2.5 h. The nowheterogeneous reaction mixture was cooled to ambient temperature and allvolatiles removed in vacuo. The crude product was then purified bychromatography on silica (30×190 mm) using 3:1 pentane/EtOAc. The mainproduct peak eluting 180-360 mL was collected, pooled and concentratedin vacuo to a colorless oil. Final purification throughrecrystallization from warm EtOAc and pentane afforded a whitecrystalline solid (0.369 g, 0.859 mmol; 85.9%).

Example 48 Syringe Adsorption of Imaging Agent 1

Three two-component syringes (Henke Sass Wolf) as well as threethree-component syringes (Becton and Dickinson) were each filled with a1 mL solution of imaging agent 1 (<5 volume % EtOH in H₂O containing <50mg/mL ascorbic acid); total initial radioactivity in each syringe wascomparable. The two sets of filled syringes were maintained at ambienttemperature and humidity for a period of three hours, at which time thecontents were injected into a clean 5 cc glass vial; a consistent volumeof imaging agent 1 (0.1 mL) remained in the hub of each syringe. Thetotal radioactivity content of both the vial and syringe were measured,decay corrected and the percent retention calculated. The values ofpercent radioactivity retained in each syringe are summarized in Table20. The difference in percent retained activity is statisticallysignificant at the 95% confidence level (i.e. Prob >|t| 0.0005).

TABLE 20 Compiled Data for imaging agent 1 Retained in Syringes SyringeRetained Activity [%] B&D 32.4 B&D 35.2 B&D 38.9 HSW 11.3 HSW 7.0 HSW5.1 B&D = Becton & Dickinson - HSW = Henke Sass Wolf

Example 49 Syringe Component Adsorption of imaging agent 1

To further identify the contact surface material that contributed tosyringe retention of imaging agent 1, three additional B&D syringes wereeach filled with a 1 mL solution of imaging agent 1 then maintained atambient temperature and humidity for a period of three hours. Followingtransfer of the individual doses as described in Example 1, the syringebarrel and butyl rubber tip plunger were then separated, measured forretained radioactivity and decay corrected. The values of percentretained radioactivity for each syringe component are summarized inTable 21. The difference in percent retained activity is statisticallysignificant at the 95% confidence level (i.e. Prob >|t| 0.0017).

TABLE 21 Percent imaging agent 1 Retained in B&D Syringe ComponentsPost-Injection Syringe Part Retained Activity [%] Plunger 30.9 Plunger27.7 Plunger 21.8 Barrel 6.9 Barrel 5.5 Barrel 7.0 B&D = Becton &Dickinson

It will be evident to one skilled in the art that the present disclosureis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element or a list of elements. In general, the term “or” as usedherein shall only be interpreted as indicating exclusive alternatives(i.e. “one or the other but not both”) when preceded by terms ofexclusivity, such as “either,” “one of,” “only one of,” or “exactly oneof.” “Consisting essentially of,” when used in the claims, shall haveits ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

What is claimed is:
 1. A method of imaging a subject, comprising:administering to a subject a first dose of imaging agent comprising theformula:

in an amount between about 1 mCi and about 4 mCi; acquiring at least onefirst image of a portion of the subject; subjecting the subject tostress; administering to the subject undergoing stress a second dose ofthe imaging agent in an amount greater than the first dose of theimaging agent by at least about 1.5 times the first dose of the imagingagent; and acquiring at least one second image of the portion of thesubject.
 2. The method of claim 1, wherein the second dose of theimaging agent is administered within less than about 48 hours, about 24hours, about 18 hours, about 12 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 30minutes, or about 15 minutes after acquiring the at least one firstimage.
 3. The method of claim 1, wherein the second dose of the imagingagent is at least about 2.0 times greater than the first dose of theimaging agent.
 4. The method of claim 1, wherein the portion of thesubject is at least a portion of the cardiovascular system.
 5. Themethod of claim 4, wherein the portion of the cardiovascular system isat least a portion of the heart.
 6. The method of claim 1, wherein theacquiring employs positron emission tomography.
 7. The method of claim1, further comprising determining the presence or absence of acardiovascular disease or condition in the subject.
 8. The method ofclaim 7, wherein the cardiovascular disease is coronary artery diseaseor myocardial ischemia.
 9. The method of claim 1, wherein the imagingagent is administered as a formulation comprising water, less than about5% ethanol, and less than about 50 mg/mL sodium ascorbate.
 10. Themethod of claim 1, wherein the stress is induced by exercising thesubject.
 11. The method of claim 10, wherein the second dose of theimaging agent is administered during the exercise.
 12. The method ofclaim 10, wherein the wait time between acquiring at least one firstimage of a portion of the subject and administering to the subject asecond dose of the imaging agent is about 60 minutes.
 13. The method ofclaim 1, wherein the second dose of the imaging agent is administered inan amount that is at least about 2.5 times greater than the first doseof the imaging agent.
 14. The method of claim 13, wherein the seconddose of the imaging agent is administered in an amount between about 2.5and about 5.0 times greater than the first dose of the imaging agent.15. The method of claim 10, wherein the second dose of the imaging agentis between about 8.6 mCi and about 9.5 mCi.
 16. The method of claim 1,wherein the stress is pharmacological stress.
 17. The method of claim16, wherein the pharmacological stress is induced by administering apharmacological stress agent to the subject.
 18. The method of claim 17,wherein the pharmacological stress agent is a vasodilator.
 19. Themethod of claim 16, wherein the second dose of the imaging agent isadministered after the subject has been administered the pharmacologicalstress agent.
 20. The method of claim 16, wherein the second dose of theimaging agent is administered when the subject is at peak vasodilationfrom the pharmacological stress agent.
 21. The method of claim 1,wherein the first dose of the imaging agent is between about 2.0 mCi toabout 3.5 mCi.
 22. The method of claim 21, wherein the second dose ofthe imaging agent is between about 5.7 mCi and about 6.2 mCi, or betweenabout 6.0 mCi and about 6.5 mCi, and about 5.7 mCi and about 6.5 mCi.23. The method of claim 1, wherein the total of the first and seconddose of the imaging agent does not exceed about 14 mCi.
 24. A method ofimaging a subject, comprising: subjecting a subject to stress;administering to the subject a first dose of an imaging agent comprisingthe formula:

in an amount between about 1 mCi and about 4 mCi; acquiring at least onefirst image of a portion of the subject; administering to the subject asecond dose of the imaging agent in an amount greater than the firstdose of the imaging agent; and acquiring at least one second image ofthe portion of the subject.
 25. The method of claim 24, wherein theamount of the second dose is more than about 1.5 times the amount of thefirst dose.